Bispecific format suitable for use in high-through-put screening

ABSTRACT

The present disclosure relates to a novel bispecific protein complex and a method of using the complexes to screen for synergistic or novel biological function. The bispecific format is particularly suitable for high-throughput screening because all of its components can be expressed from cells as individual units and the units can be assembled simply by mixing without employing conjugation or coupling chemistry.

FIELD OF INVENTION

The present disclosure relates to a method, in particular an in vitro/exvivo method, of detecting synergistic biological function in aheterodimerically-tethered bispecific protein complex,libraries/multiplexes of the bispecific protein complexes, and kits andcompositions thereof. The disclosure further relates to said novelbispecific protein complexes and use of the same in therapy, researchand experimental purposes (in particular in assays looking forsynergistic biological function). The present disclosure also extends tomethods of preparing said bispecific complexes.

BACKGROUND OF INVENTION

Biological mechanisms in vivo are extremely complicated cascades ofsignals, which are difficult to deconvolute and understand. An exampleof such signalling is that required to activate T-cells, see FIG. 1,from www.cellsignal.com. Activation of T cells requires at least twosignals.

The recognition of the antigen by the T cell receptor is considered thefirst signal and the second signal arises from co-stimulation whichresults from the ligation of additional surface molecules on the T cellwith additional molecules on an antigen presenting cell.

Thus T cell activation can be used to illustrate that the modulation ofbiological functions can require multiple signals. Other biologicalprocesses are equally complicated or more complicated. Whilst in vitroscreening based on cells has and can assist with gaining insights intoin vivo mechanisms the problem still arises of how to identifyappropriate ligand pairs which modulate the biological function.

Bispecific antibodies are widely expected to play a major role in thenext generation of biotherapeutics (D. Holmes, Nature Rev Drug DiscNovember 2011:10; 798). They have the potential to deliver superior,long term, broad efficacy in a greater proportion of patients. This canbe achieved by either co-engaging different antigens simultaneouslywithin a common disease pathway, thereby reducing redundancy; or bytargeting antigens from independent pathways to provide an additive orsynergistic effect.

Bispecific antibodies facilitate access to novel biology such as:

-   -   1) cross-linking receptors on a cell,    -   2) inducing cell mediated effects,    -   3) localizing a cytokine to a cell to regulate signaling or        locally block cytokine function,    -   4) engaging multiple epitopes simultaneously to generate “new        activity”, increase function or specificity, which may not be        exhibited by a single monoclonal antibody or indeed mixtures of        un-linked antibodies (‘poly-monoclonals’).

Current strategies to engage dual targets are largely based on rationaldesign of known mechanisms and include: cross-linking inhibitoryreceptors, co-engagement/clustering of receptors, blocking multiplestimulatory pathways, selective engagement of inhibitory receptors andblocking distinct pathways such as co-stimulation & cytokine signaling.However, the current state of the art in relation to known mechanismsand targets is a limiting factor to progress in this area.

Whilst bispecific antibodies have enormous potential as biologicaltherapeutics they also present an increased set of challenges withindiscovery and development compared to monoclonal antibodies. Two keyareas of difficulty are, 1) the development of a successful bispecificantibody format, and 2) selecting the pairs of targets to which thebispecific antibody will crosslink or co-engage.

Many promising bispecific antibody formats have now been developed thatcould potentially work as successful therapeutics including DVD-Ig(Abbvie), DuoBodies (Genmab), Knobs-in-Holes (Genentech), Common lightchain (Merus). However, in each of these cases these formats are notideally suited to high throughput target-dual-antigen discoveryscreening to enable the discovery of novel antigen pairs forcrosslinking with bispecific antibodies.

Typically for a single bispecific antibody construct at least twovariable regions need to be sub-cloned from the original source ofdiscovery vectors (e.g. phage display, hybridoma or single B-cellcloning) into appropriate bispecific expression vectors, each arm of thebispecific has to be expressed and the resulting bispecific antibodypurified. This cloning and subsequent expression effort quickly becomesa significant practical bottleneck if large numbers of pairs of variableregions are to be combined in an attempt to screen for the mostefficacious combination of discovered variable regions or to discovernovel antigen pairs.

For example, if 50 unique antibodies are discovered against a panel of50 cell surface targets, then a total of 2500 bispecific antibodiescould potentially be generated (envisaged as an X-by-Y grid). With thebispecific antibody formats described above this would require at least100 individual cloning reactions (50-X and 50-Y) followed by 2500antibody expression experiments. Increasing the number of startingmonoclonal antibodies to 100 would increase the minimal number ofcloning reactions to 200 (100-X and 100-Y) and the expression number to10,000.

Generally the root cause of this ‘expression bottleneck’ is the factthat the formats described above require both protein chain ‘halves’ ofthe final bispecific construct to be expressed simultaneously within asingle expression experiment in the same cell. Therefore, for manyformats, to produce 2500 bispecific antibodies, 2500 expressionexperiments are required.

The ‘expression bottleneck’ is further exacerbated if the bispecificantibody format is monocistronic (i.e. cloned and expressed as a singlechain protein), for example single chain diabodies, as the number ofcloning experiments would be 2500 and 10,000 respectively for thenumbers given above.

Furthermore after expression, extensive purification may be required toisolate the desired construct.

Some bispecific approaches employ a common light chain in the bispecificconstructs in order to reduce the amount of cloning, although thisdoesn't reduce the number of expression experiments. Furthermore, usinga common chain, such as a common light chain, makes the challenge ofantibody discovery harder as it is more difficult to find the startingantibody variable domains as the antibody needs to bind its antigen witha high enough affinity through one chain, such as the heavy chain,alone.

Accordingly the use of current bispecific formats in large scale andhigh throughput screening to identify novel antigen pairs is impracticaland has led to the continued use of solely hypothesis driven approachesto bispecific antigen targeting.

We propose that rather than designing and testing a limited selection ofbispecific antibodies that engage given epitopes on two known targets,the true potential of exploiting access to novel biology with bispecificantibodies can only be achieved through a broad functional screeningeffort with a large, diverse combinatorial panel of bispecificantibodies or protein ligands. To facilitate this screening a format anda method is required that enables the generation of large numbers ofdiverse bispecific proteins which can be readily constructed andscreened for functional effects in a variety of functional screens. Thisapproach allows for the serendipitous identification of synergisticpairs.

Thus it would be useful to generate and screen a large number ofbispecific protein complexes present as combinations of various antigenspecificities. In particular, it would be useful to be able to generateand screen a large number of different bispecific antibody complexes ina quick and efficient manner. There are a range of existing methods formanufacturing bispecific antibodies as already described above. However,each of these methods has its disadvantages, as do alternative methodsas further described in more detail below.

The problem of how to efficiently identify targets for bispecific andmultispecific constructs has not been adequately addressed in the art.For example WO2014/001326 employs chemical conjugation of a protein to aDNA fragment, wherein the DNA fragment hybridises to a complementary DNAsequence that links two such proteins together for generatingtailor-made patient-specific multispecific molecules comprising at leasttwo targeting entities. There are number of difficulties associated withthis approach if it were to be applied to identifying new bispecificcombinations, for example conjugation of the protein to the DNA canresult in damage to the activity and/or structure of the protein. Inparticular protein-DNA hybrids are not naturally occurring thus there isa potential for interference. In addition the chemical conjugationrequired to join the protein and DNA adds complexity, time and expenseto the process.

Coupling and conjugation techniques exist for generating antibody drugconjugates and in vivo targeting technologies. Traditional chemicalcross-linking is labour intensive as the relevant species may need to bepurified from homodimers and other undesirable by-products. In addition,the chemical modification steps can alter the integrity of the proteins,thus leading to poor stability or altered biological function. As aresult, the production of bispecific antibodies by chemicalcross-linking is often inefficient and can also lead to a loss ofantibody activity.

Another method of manufacturing bispecific antibodies is by cell-fusion(e.g. hybrid hybridomas), wherein the engineered cells express two heavyand two light antibody chains that assemble randomly. Since there are 4possible variants to choose from, this results in the generation of 10possible bispecific antibody combinations, of which only some (in manycases, only one) combinations would be desired. Hence, generatingbispecific antibodies by cell-fusion results in low production yieldsand also requires an additional purification step in order to isolatethe desired bispecific antibodies from the other bispecific antibodiesproduced. These disadvantages increase manufacturing time and costs.

Recombinant DNA techniques have also been employed for generatingbispecific antibodies. For example, recombinant DNA techniques have alsobeen used to generate ‘knob into hole’ bispecific antibodies. The ‘knobinto hole’ technique involves engineering sterically complementarymutations in multimerization domains at the CH3 domain interface (seee.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al.,Nat. Biotechnol. 16(7): 677-81 (1998); see also U.S. Pat. Nos. 5,731,168and 7,183,076). One constraint of this strategy is that the light chainsof the two parent antibodies have to be identical to prevent mispairingand formation of undesired and/or inactive molecules when expressed inthe same cell. Each bispecific (heavy and light chains thereof) must beexpressed in a single cell and the protein product generally containsabout 20% of homodimer, which is subsequently removed by purification.

Other approaches are based on the natural exchange of chains infull-length IgG4 molecules (Genmab Dubody). However, this approach alsohas difficulties because it does not allow a construct to be preparedwithout an Fc region. As the Fc region can contribute to biologicalactivity it may be difficult to establish if an activity observed isbased on the combination of variable regions, the Fc or both inbispecific molecules comprising an Fc. Furthermore, the exchange is adynamic process and this may lead to difficulties in relation to whatthe entity tested actually is.

Thus there is a need for new methods of generating bispecific proteincomplexes to enable the more efficient and higher throughput screeningof bispecific antibodies. In particular, there is a need for a formatand a method wherein a selection of any two antibodies or antibodyfragments from a pool of available antibodies or antibody fragments canbe readily combined to efficiently produce a multiplex of differentbispecific antibodies, whilst, for example avoiding or minimising theformation of homodimers. Assembling different bispecific antibodiesefficiently is particularly important when screening for synergisticbiological function for new combinations of antigen specificities, inparticular where heterodimers are essential for discovering thatfunction.

SUMMARY OF INVENTION

In one aspect there is provided a new bispecific format particularlysuitable for use in screening because all of the components can beexpressed from a cell as individual units, essentially withoutaggregation and the units can be assembled simply by mixing withoutemploying conjugation or coupling chemistry and with minimalhomodimerisation.

Thus there is provided a bispecific protein complex having the formulaA-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   : is a binding interaction between X and Y;    -   A is a first protein component of the bispecific protein complex        selected from a Fab or Fab′ fragment;    -   B is a second protein component of the bispecific protein        complex selected from a Fab or Fab′ fragment;    -   X is a first binding partner of a binding pair independently        selected from an antigen or an antibody or a binding fragment        thereof; and    -   Y is a second binding partner of the binding pair independently        selected from an antigen or an antibody or a binding fragment        thereof;

with the proviso that when X is an antigen Y is an antibody or bindingfragment thereof specific to the antigen represented by X and when Y isan antigen X is an antibody or binding fragment thereof specific to theantigen represented by Y.

In one embodiment the variable X or Y is an antibody binding fragmentsuch as a scFv, Fv, VH, VL or VHH and the other variable is a peptide.

In one embodiment the variable X or Y is a scFv or VHH and the othervariable is a peptide. Thus the bispecific format comprises two Fab armswith different specificities linked, for example via their C-terminal,by an antibody binding fragment (such as a scFv or a VHH) and peptidebinding interaction. This type of arrangement is ideal for use inscreening because there is no difficulty expressing the unit A-X or theunit B-Y. The Fab/Fab′ fragment is very stable and is not susceptible toinappropriate dimerization. Thus the amount of purification requiredafter expression of each unit (A-X or B-Y) is minimal or in fact,unnecessary. The bispecific complex can be formed in a 1:1 molar ratioby simply admixing the relevant units i.e. without recourse toconjugation and coupling chemistry. The constant regions in the Fab/Fab′fragment drive dimerization of the Fab/Fab′ components and the bindingpartners X and Y drive the equilibrium further in favour of forming therequisite heterodimer bispecific complex. Again little or nopurification is required after formation of the complex afterheterodimerisation. Thus large number of A-X and B-Y can be readilyprepared and combined.

The Fab/Fab′ entities in the complex mean the binding domains are heldin biologically relevant orientations which mimic classic antibodygeometry and this may contribute to the success of translating the pairsof variable regions identified by the screening method described hereinbelow into other bispecific therapeutic formats which retain activity.The ability to prepare and screen a bispecific complex lacking the Fcfragment CH2-CH3 also ensures that the biological activity observed isin fact due solely to the variable region pairs in the complex. Thesimplicity of the bispecific complex of the invention and the methods ofpreparing it are a huge advantage in the context of facilitatinghigh-through-put screening of variable domain pairs to find new targetantigen combinations and also to optimise variable region sequences fora given combination.

In one embodiment A is a Fab fragment. In one embodiment B is a Fabfragment. In one embodiment A and B are both a Fab fragment (alsoreferred to herein as a Fab-Kd-Fab).

In one embodiment X is fused to the C-terminal of a heavy chain or lightchain in the Fab or Fab′ fragment, in particular the C-terminal of theheavy chain.

In one embodiment Y is fused to the C-terminal of a heavy chain or lightchain in the Fab or Fab′ fragment, in particular the C-terminal of theheavy chain.

In one embodiment X is independently selected from a scFv, a VHH and apeptide, with the proviso that when X is a peptide Y is an antibody orbinding fragment thereof, such as a scFv or VHH and when X is a scFv orVHH then Y is an antigen, such as a peptide.

In one embodiment Y is independently selected from a scFv, a VHH and apeptide, with the proviso that Y is a peptide X is an antibody orbinding fragment, such as a scFv or VHH and when Y is a scFv or a VHHthen X is an antigen, such as a peptide.

In one embodiment the peptide (which is one of the binding partners) isin the range 5 to 25 amino acids in length.

In one embodiment the binding affinity between X and Y is 5 nM orstronger, for example the binding affinity of the heterodimeric tetheris 900 pM or stronger, such as 800, 700, 600, 500, 400 or 300 pM.

In one embodiment X or Y is a scFv or VHH specific to the peptide GCN4,for example the scFv is 52SR4 (SEQ ID NO:3 or amino acids 1-243 of SEQID NO:3).

In one embodiment X or Y is a peptide GCN4 (SEQ ID NO:1 or amino acids1-38 of SEQ ID NO:1).

In one embodiment A and/or B is specific for an antigen selected fromthe group comprising: cell surface receptors such as T cell or B cellsignalling receptors, co-stimulatory molecules, checkpoint inhibitors,natural killer cell receptors, Immunolglobulin receptors,immunoglobulin-like receptors, matrix metalloproteases and membrane typematrix metalloproteases tissue inhibitors of metalloproteases, TNFRfamily receptors, B7 family receptors, adhesion molecules, integrins,cytokine/chemokine receptors, GPCRs, growth factor receptors, kinasereceptors, tissue-specific antigens, cancer antigens (tumour associatedantigens & peptides), pathogen recognition receptors, complementreceptors, hormone receptors, scavenger receptors, or soluble moleculessuch as cytokines, chemokines, leukotrienes, growth factors, hormones orenzymes or ion channels, including post translationally modified versionthereof, fragments thereof comprising at least one epitope.

In one embodiment there is provided a composition, for example apharmaceutical composition comprising one or more bispecific complexesaccording to the present disclosure.

Furthermore, the present inventors have devised a method of detectingsynergistic function in a heterodimerically-tethered bispecific proteincomplex of formula A-X:Y-B wherein X:Y is a heterodimeric-tether, forexample where X and Y are unsuitable for forming homodimers,

A and B are components of the bispecific in the form of fusion proteinswith X and Y respectively, said method comprising the steps of:

-   -   (i) testing for activity in a functional assay for part or all        of a multiplex comprising at least one        heterodimerically-tethered bispecific protein; and    -   (ii) analysing the readouts from the functional assay to        identify synergistic biological function in the bispecific        protein complex.

The method employs a novel bispecific protein complex format having thefollowing formula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   A is a first protein component of the bispecific;    -   B is a second protein component of the bispecific;    -   X is a first binding partner of a binding pair;    -   Y is a second binding partner of the binding pair; and    -   : is an interaction (for example a binding interaction) between        X and Y, in particular the interaction is sufficient to form the        complex and retain the fusion proteins in a complexed form.

In particular, the heterodimerically-tethered bispecific protein complexis prepared by mixing A-X and B-Y in vitro. Thus in one embodiment themethod comprises an in vitro mixing step bringing A-X and B-Y intocontact.

Thus generally the fusion proteins A-X and B-Y are not co-expressed inthe same cell. This is advantageous because it allows, for example 100fusion proteins to expressed and optionally purified and the subsequentmixing of the 100 fusion proteins in the various permutations canprovide 10,000 heterodimerically-tethered bispecific protein complexes,of which 5,000 are unique pairs.

In contrast certain prior art methods require co-expression ofbispecifics and thus for 10,000 complexes, 10,000 transfections,expressions and purifications are required.

However, if desired the A-X and B-Y may be expressed in the same cell.

The binding partners X and Y have affinity for each other and act asbiological equivalent of VELCRO® or a bar and magnet and hold thecomplex together. Advantageously, this means that the fusion proteinsA-X and Y-B can be readily assembled into a bispecific protein complexsimply by mixing the fusion proteins together. Thus the bispecificprotein complex of the present disclosure has a modular structure whichallows for two different proteins to be easily assembled in order toproduce large panels of permutations of bispecific protein complexeswith different combinations of antigen specificities in, for example agrid-like fashion. This allows for the efficient and systematicscreening of a large number of bispecific protein complexes in order todetect additive, synergistic or novel biological function.

Given X and Y are specific for each other this significantly reduces theability to form homodimers. X and Y are collectively referred to hereinas a binding pair or binding partners. In one embodiment X does not havehigh affinity for other Xs. In one embodiment Y does not have highaffinity for other Ys. Advantageously, when X and Y do not formhomodimers, this prevents the formation of undesired monospecificprotein complexes, increases yield of the desired bispecific proteincomplexes, and removes the need for onerous purification steps to removethe monospecific protein complexes.

This allows rapid assembly of bispecific protein complexes with a yieldand/or purity which cannot be obtained efficiently by most prior artmethods, in particular prior art methods generally require extensivepurification steps. The yield of bispecific complex is typically 75% orhigher in the present invention.

Further advantageously, the bispecific protein complexes allow for thescreening of complexes wherein the constituent proteins (includingantigens bound by the constituent proteins) do not have a knownrelationship or are in different potentially unrelated pathways, suchas, two proteins which function in two distinct pathways and, forexample which the skilled person would not normally expect to come intocontact with each other can be tested in a bispecific protein complex toidentify additive, synergistic and/or novel function.

Furthermore multiple binding regions (such as variable regions) to agiven antigen or epitope can be investigated in parallel to identifynuances in biological function. This allows combinations of variableregion sequences directed to a given pair of antigens to be investigatedand optimised.

The present method allows the science to show the results and does notrely on pre-conceived ideas and technical prejudice about the biologicalfunction. This approach is potentially very powerful.

Advantageously the X and Y components allow a multiplex comprisingbispecific protein complexes made up of different permutations of fusionproteins to be assembled rapidly and easily.

In one embodiment the proteins A and B are antibodies or antibodyfragments. When the antibody or antibody fragments are held together asa complex via X and Y, this forms a bispecific antibody complex.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the main cell signalling pathwaysinvolved in the activation of T cells.

FIG. 2 is a schematic diagram showing the structure and assembly of abispecific protein complex of the present disclosure.

FIG. 3 is a table showing an example 4×4 grid for functional screeningusing the bispecific antibody of the present invention. Using this grid,16 different bispecific protein complexes can be assembled andefficiently screened for synergistic function.

FIG. 4 is a schematic diagram showing a representative bispecificantibody complex of the present disclosure. The diagram depicts how twodifferent Fab fragments come together to form a bispecific antibodycomplex via the high affinity interaction between the binding partnersattached to the Fab fragments.

FIG. 5 is a graph showing the results of a flow cytometry experimentwhich demonstrates that two Fab fragments which are specific for twodifferent target antigens retain their specificities and are able toco-engage their corresponding target antigens simultaneously when thetwo Fab fragments are combined to form a bispecific antibody complex ofthe present disclosure. The results further demonstrate that reversingthe attachment of the binding partners attached to the Fab fragmentsdoes not affect the ability of the Fab fragments to bind specifically totheir respective target antigens. The no complex formation control showsno binding detected when both specificities are fused to peptide (Y:Y).

Filled area=[anti-antigen 5 Fab-peptide ‘GCN4’]:[anti-antigen 6Fab-peptide ‘GCN4’]:[biotinylated-antigen 6]:[FITC-STREP]. No complexformation control.

Thin line=[anti-antigen 5 Fab-scFv ‘52SR4’]:[anti-antigen 6 Fab-peptide‘GCN4’]:[biotinylated-antigen 6]:[FITC-STREP]

Thick line=[anti-antigen 5 Fab-peptide ‘GCN4’]:[anti-antigen 6 Fab-scFv‘52SR4’]:[biotinylated-antigen 6]:[FITC-STREP]

FIG. 6 is a graph and a table showing a BIAcore trace, whichdemonstrates the high affinity of the binding partners for each other.Fab A-scFv ‘52SR4’ binding is detected to peptide ‘GCN4’ on the chip.

FIG. 7 is a bar chart of the relative potency of inhibition ofphosphorylated Akt for bispecific and bivalent combinations ofantibodies with specificity for antigen 3, antigen 1, antigen 4 andantigen 2.

FIG. 8 is a bar chart of the relative potency of inhibition ofphosphorylated PLCg2 for bispecific and bivalent combinations ofantibodies with specificity for antigen 3, antigen 1, antigen 4 andantigen 2.

FIG. 9 is a bar chart of the relative potency of inhibition of CD86expression for bispecific and bivalent combinations of antibodies withspecificity for antigen 3, antigen 1, antigen 4 and antigen 2.

FIG. 10 is a bar chart of the relative potency of inhibition ofphosphorylated Akt for bispecific, bivalent or mixtures of antibodieswith specificity for antigen 1 and antigen 2 as well as single Fab′controls.

FIG. 11 is a bar chart of the relative potency of inhibition ofphosphorylated Akt for bispecific, bivalent or mixtures of antibodieswith specificity for antigen 3 and antigen 2.

FIG. 12 is a bar chart of the relative potency of inhibition ofphosphorylated PLCg2 for bispecific, bivalent or mixtures of antibodieswith specificity for antigen 3 and antigen 2.

FIG. 13 is a graph showing the titration of the effect of the bispecificcombination of anti-antigen 3 and anti-antigen 2 on total IkB levels inanti-IgM stimulated B cells.

FIG. 14 is a graph showing the titration of the effect of the bispecificcombination of antigen 3 and antigen 2 on CD86 expression on anti-IgMstimulated B cells.

FIG. 15 is a bar chart of the relative potency of inhibition ofphosphorylated Akt for bispecific, bivalent or mixtures of antibodieswith specificity for antigen 4 and antigen 2.

FIG. 16 is a bar chart of the relative potency of inhibition ofphosphorylated PLCg2 for bispecific, bivalent or mixtures of antibodieswith specificity for antigen 4 and antigen 2.

FIG. 17 is a graph showing the titration of the effect of the bispecificcombination of antigen 4 and antigen 2 on CD86 expression on anti-IgMstimulated B cells.

FIG. 18 is a graph showing the overlaid size exclusion A280 signaltraces from experiment 1 of Example 11. The traces shown are: Fab-X(VR4247) control, Fab-Y (VR4248) control and 1 to 1 molar ratio mixtureat 500 μg/ml of Fab-X (VR4247) and Fab-Y (VR4248) complex. Peaks weredetected at an absorbance of 280 nm.

FIG. 19 is a graph showing the overlaid size exclusion A214 signaltraces from experiment 2 of Example 11. The traces shown are: Fab-X(VR4130) control, Fab-Y (VR4131) control and 1 to 1 molar ratio mixtureat 500 μg/ml of Fab-X (VR4130) and Fab-Y (VR4131) complex. Peaks weredetected at an absorbance of 214 nm.

FIG. 20 is a graph showing the overlaid size exclusion A214 signaltraces from experiment 2 of Example 11. The traces shown are all Fab-X(VR4130)/Fab-Y (VR4131) 1 to 1 molar ratio mixtures at 500 μg/ml, 50μg/ml and 5 μg/ml as indicated. Peaks were detected at an absorbance of214 nm.

FIG. 21 is a table showing the data for the antigen grid crossspecificities. Values are percentage inhibition (negative value foractivation) of phosphorlylation of Syk & represent the mean of multipleV region combinations evaluated.

FIG. 22 is a table showing the data for the antigen grid crossspecificities. Values are percentage inhibition (negative value foractivation) of phosphorlylation of PLCg2 & represent the mean ofmultiple V-region combinations evaluated.

FIG. 23 is a table showing the data for the antigen grid crossspecificities. Values are percentage inhibition (negative value foractivation) of phosphorlylation of AKT & represent the mean of multipleV region combinations evaluated.

FIG. 24 is a graph showing the percentage inhibition of thephosphorlylation of Syk, PLCg2 & AKT for each V-region combination forantigen 2 in Fab-X combined with antigen 3 in Fab-Y

FIG. 25 is a graph showing the percentage inhibition of thephosphorlylation of Syk, PLCg2 & AKT of the phosphorlylation of Syk,PLCg2 & AKT for each V-region combination for antigen 3 in Fab-Xcombined with antigen 2 in Fab-Y.

FIG. 26 is a graph showing the percentage inhibition of thephosphorlylation of Syk, PLCg2 & AKT for each V region combination forantigen 2 in Fab-X combined with antigen 4 in Fab-Y.

FIG. 27 is a graph showing the percentage inhibition of thephosphorlylation of Syk, PLCg2 & AKT for each V region combination forantigen 4 in Fab-X combined with antigen 2 in Fab-Y.

FIG. 28 is a graph showing the data for the percentage inhibition ofanti-IgM induced CD71 expression on B-cells, by antigen3Fab′-X andantigen2-Fab′-Y when combination as either purified Fab′ or fromtransient supernatant.

Circles—Purified Antigen2Fab-Y+Antigen3Fab-X IC₅₀ 0.3224 nM

Squares—Transient Sup Antigen2-Y+Antigen3-X IC₅₀ 0.2640 nM

Triangle—Mock transfected supernatant control

FIG. 29 is a graph showing the data for the percentage inhibition ofanti-IgM induced phosphorylation of p38 in B-cells, by antigen3-Fab′-Xand antigen 2-Fab′-Y when combination as either purified Fab′ or fromtransient supernatant.

Circles—Purified Antigen2Fab-Y+Antigen3Fab-X IC₅₀ 0.1413 nM

Squares—Transient Sup Antigen 2-Y+Antigen 3-X IC₅₀ 0.1861 nM

Triangle—Mock transfected supernatant control

Key for FIGS. 30 to 33

1. Antigen2Fab-Y (VR4447)+Antigen3Fab-X (VR6066); 2. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6078); 3. Antigen2Fab-Y (VR4447)+Antigen3Fab-X(VR6079); 4. Antigen2Fab-Y (VR4447)+Antigen3Fab-X (VR6080); 5.Antigen2Fab-Y (VR4447)+Antigen3Fab-X (VR6082); 6. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6067); 7. Antigen2Fab-Y (VR4447)+Antigen3Fab-X(VR6068); 8. Antigen2Fab-Y (VR4447)+Antigen3Fab-X (VR6070); 9.Antigen2Fab-Y (VR4447)+Antigen3Fab-X (VR6071); 10. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6073); 11. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6075); 12. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6076); 13. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6077); 14. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6069); 15. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6072); 16. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6074); 17. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (VR6081); 18. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (TSUP-24117); 19. Antigen2Fab-Y(VR4447)+Antigen3Fab-X (TSUP-24432); 20. Mock supe 1; 21. Mock supe 2;22. Antigen2Fab-Y (VR4447)+Antigen3Fab-X (4126) purified

FIG. 30 is a graph showing the inhibition of phospho readouts bypurified antigen2-specific Fab-Y (VR4447) combined withantigen3-specific Fab-X transients on IgM stimulated B-cells fromUCB_Cone_172.

FIG. 31 is a graph showing the inhibition of phosphor readouts bypurified antigen 2-specific Fab-Y (VR4447) combined with antigen3-specific Fab-X transients on IgM stimulated B-cells from UCB_Cone_173

FIG. 32 Inhibition of phospho readouts by purified antigen 2-specificFab-Y (VR4450) combined with antigen 3-specific Fab-X transients on IgMstimulated B-cells from UCB_Cone_172

FIG. 33 Inhibition of phospho readouts by purified antigen 2-specificFab-Y (VR4450) combined with antigen 3-specific Fab-X transients on IgMstimulated B-cells from UCB_Cone_173

FIG. 34 shows data for the percentage inhibition of anti-IgM inducedphosphorylated PLCy2 in B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe

FIG. 35 shows data for the percentage inhibition of anti-IgM inducedphosphorylated P38 in B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe

FIG. 36 shows data for the percentage inhibition of anti-IgM inducedphosphorylated Akt in B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe

FIG. 37 shows data for the percentage inhibition of anti-IgM inducedCD71 expression on B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe

FIG. 38 shows data for the percentage inhibition of anti-IgM inducedCD40 expression on B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe.

FIG. 39 shows data for the percentage inhibition of anti-IgM inducedCD86 expression on B-cells, by antigen 3 and antigen 2 specificFab-Kd-Fab or BYbe

FIG. 40 shows the inhibition of CD27 expression on B cells byVR4447/VR4126 BYbe and VR4447/VR4126/VR645 BYbe/Albumin

FIG. 41 shows the inhibition of CD71 expression on B cells byVR4447/VR4126 BYbe and VR4447/VR4126/VR645 BYbe/Albumin

FIG. 42 shows the inhibition of CD86 expression on B cells byVR4447/VR4126 BYbe and VR4447/VR4126/VR645 BYbe/Albumin

FIG. 43 shows the inhibition of CD27 expression on B cells byVR4447/VR4130 BYbe and VR4447/VR4130/VR645 BYbe/Albumin

FIG. 44 shows the inhibition of CD71 expression on B cells byVR4447/VR4130 BYbe and VR4447/VR4130/VR645 BYbe/Albumin

FIG. 45 shows the inhibition of CD86 expression on B cells byVR4447/VR4130 BYbe and VR4447/VR4130/VR645 BYbe/Albumin

DETAILED DESCRIPTION

“Bispecific protein complex” as used herein refers to a moleculecomprising two proteins (A and B referred to herein as bispecificcomponents also referred to herein as the first protein component andsecond protein component, respectively of the bispecific) which areretained together by a heterodimeric-tether. In one embodiment one orboth of the proteins have a binding domain, for example one or both ofthe proteins are antibodies or fragments thereof (in particular a Fab orFab′ fragment, such complexes are also referred to as Fab-Kd-Fab).“Fusion proteins” as employed herein comprise a protein component A or Bfused to a binding partner X or Y (as appropriate). In one embodimentthe fusion protein is a translational protein expressed by recombinanttechniques from a genetic construct, for example expressed in a hostfrom a DNA construct. In the context of the present disclosure one ofthe key characteristics of a fusion protein is that it can be expressedas a “single protein/unit” from a cell (of course in the case of fusionproteins comprising a Fab/Fab′ fragment there will be two chains butthis will be considered a single protein for the purpose of the presentspecification with one chain, typically the heavy chain fused at itsC-terminus to X or Y as appropriate, optionally via a linker asdescribed herein below).

The function of the heterodimeric tether X:Y is to retain the proteins Aand B in proximity to each other so that synergistic function of A and Bcan be effected or identified, for example employing the methoddescribed herein.

“heterodimeric-tether” as used herein refers to a tether comprising twodifferent binding partners X and Y which form an interaction:(such as abinding) between each other which has an overall affinity that issufficient to retain the two binding partners together. In oneembodiment X and/or Y are unsuitable for forming homodimers.

Heterodimerically-tethered and heterodimeric-tether are usedinterchangeably herein.

In one embodiment “unsuitable for forming homodimers” as employed hereinrefers to formation of the heterodimers of X-Y are more preferable, forexample more stable, such as thermodynamically stable, once formed thanhomodimers. In one embodiment the binding interaction between X and Y ismonovalent.

In one embodiment the X-Y interaction is more favourable than the X-X orY-Y interaction. This reduces the formation of homodimers X-X or Y-Ywhen the fusion proteins A-X and B—Y are mixed. Typically greater than75% heterodimer is formed following 1:1 molar ratio mixing.

If desired, a purification step (in particular a one-step purification),such as column chromatography may be employed, for example to purify thefusion protein units and/or bispecific protein complexes according tothe present disclosure.

In one embodiment a purification step is provided after expression ofthe or each fusion protein, although typically aggregate levels are low.Thus in one embodiment prior to in vitro mixing, the fusion protein(s)is/are provided in substantially pure form. Substantially pure form asemployed herein refers to wherein the fusion protein is 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100% monomer.

In one embodiment no purification of the fusion protein or proteins isperformed.

In one embodiment each fusion protein unit is expressed in a differentexpression experiment/run.

In one embodiment no purification of the fusion protein or proteins isperformed before mixing to generate a bispecific protein complex. In oneembodiment no purification of the fusion protein or proteins isperformed before and/or after mixing.

In one embodiment no purification is required after the bispecificprotein complex formation. In one embodiment after mixing, and generallywithout further purification, at least 50% of the composition is thedesired bispecific protein complex, for example at least 60, 65, 70, 75,80% of the composition is the required bispecific protein complex.

In one embodiment the ratio of fusion proteins employed in the in vitromixing step of the present method is A-X to B-Y 0.8:1 to 3:1, such as1.5:1 or 2:1.

In one embodiment the ratio of fusion proteins employed in the in vitromixing step of the present method is B-Y to A-X 0.8:1 to 3:1, such as1.5:1 or 2:1, in a particular a molar ratio.

In one embodiment the ratio of A-X to B-Y employed in the in vitromixing step is 1:1, in particular a 1:1 molar ratio.

The present disclosure also extends to a method of preparing abispecific complex according to the present disclosure comprisingadmixing a fusion protein A-X and B-Y, for example in a 1:1 molar ratio.

In one embodiment the mixing occurs in vitro.

In one embodiment mixing occurs in a cell, for example a host cell.

In one embodiment, the mixing occurs in vivo, i.e. the fusion proteinsA-X and B-Y interact with each other within a subject's body to form theheterodimeric-tether and in consequence, the bispecific protein complex.

In one embodiment, X and Y are completely specific for each other and donot bind to any other peptides/proteins in a cell or within a subject'sbody. This can be achieved for example by ensuring that X and Y are notnaturally present in the target cell or in the target subject's body.This can be achieved, for example by selecting X or Y to be from aspecies or entity which is different to the subject (e.g. a yeastprotein) and ensuring the other variable is specific to it.Advantageously, this prevents the binding of the fusion proteins A-Xand/or B-Y to an undesired target, thereby generating unwantedoff-target effects.

In one embodiment one (or at least one) of the binding partners isincapable of forming a homodimer, for example an amino acid sequence ofthe binding partner is mutated to eliminate or minimise the formation ofhomodimers.

In one embodiment both of the binding partners are incapable of forminga homodimer, for example an amino acid sequence of the peptide bindingpartner is mutated to eliminate or minimise the formation of homodimersand a VHH specific thereto is employed.

Incapable of forming homodimers or aggregates as employed herein, refersto a low or zero propensity to form homodimers or aggregate. Low asemployed herein refers to 5% or less, such as 4, 3, 2, 1, 0.5% or lessaggregate, for example after mixing or expression or purification.

Small amounts of aggregate in the fusion proteins or residual in theheterodimerically-tethered bispecific protein complex generally hasminimal effect on the screening method of the present disclosure.Therefore, in one embodiment no purification of fusion protein(s) and/orbispecific protein complex(es) is/are employed in the method, inparticular after the mixing step.

In one embodiment : is a binding interaction based on attractive forces,for example Van der Waals forces, such as hydrogen bonding andelectrostatic interactions, in particular, based on antibody specificityfor an antigen (such as a peptide).

In one embodiment : is a covalent bond formed from a specific chemicalinteraction, such as click chemistry. In one embodiment : is not acovalent bond. In one embodiment conjugation/coupling chemistry is notemployed to prepare the bispecific protein complexes of the presentdisclosure.

“Form the complex” as employed herein refers to an interaction,including a binding interaction or a chemical reaction, which issufficiently specific and strong when the fusion protein components A-Xand B-Y are brought into contact under appropriate conditions that thecomplex is assembled and the fusion proteins are retained together.

“Retained together” as employed herein refers to the holding of thecomponents (the fusion proteins) in the proximity of each other, suchthat after X:Y binding the complex can be handled as if it were onemolecule, and in many instances behaves and acts like a single molecule.In one embodiment the retention renders the complex suitable for use inthe method disclosed herein, i.e. suitable for use in at least onefunctional screen.

Specificity as employed herein refers to where, for example the partnersin the interaction e.g. X:Y or A and antigen or B and antigen onlyrecognise each other or have significantly higher affinity for eachother in comparison to non-partners, for example at least 2, 3, 4, 5, 6,7, 8, 9, 10 times higher affinity, than for example a background levelof binding to an unrelated non partner protein.

Specificity in relation to X and Y as employed herein refers to wherethe binding partners X and Y in the interaction only recognise eachother or have significantly higher affinity for each other in comparisonto non-partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 timeshigher affinity.

In one embodiment the binding interaction is reversible. In oneembodiment the binding interaction is essentially irreversible.

Essentially irreversible as employed herein refers to a slow off rate(dissociation constant) of the antibody or binding fragment.

In one embodiment, the binding interaction between X and Y has a lowdissociation constant. Examples of a low dissociation constant include1-9×10⁻² s⁻¹ or less, for example 1-9×10⁻³ s⁻¹, 1-9×10⁻⁴ s⁻¹, 1-9×10⁻⁵s⁻¹, 1-9×10⁻⁶ s⁻¹ or 1-9×10⁻⁷ s⁻¹. Particularly suitable dissociationconstants include 2×10⁻⁴ s⁻¹ or less, for example 1×10⁻⁵ s⁻¹, 1×10⁻⁶ s⁻¹or 1×10⁻⁷ s⁻¹.

Whilst not wishing to be bound by theory it is thought that the lowdissociation constant (also referred to as off rate) allows themolecules to be sufficiently stable to render the bispecific proteincomplex useful, in particular in functional screening assays.

In one embodiment, the affinity of X and Y for each other is 5 nM orstronger, for example 4 nM, 3 nM, 2 nM, 1 nM or stronger.

In one embodiment, the affinity of X and Y for each other is 900 pM orstronger, such as 800, 700, 600, 500, 400, 300, 200, 100 or 50 pM orstronger.

In another embodiment, the affinity of X and Y for each other is 10 pMor stronger, for example 9, 8, 7, 6 or 5 pM.

Affinity is a value calculated from the on and off rate of the entity.The term “affinity” as used herein refers to the strength of the sumtotal of non-covalent interactions between a single binding site of amolecule (e.g. an antibody) and its binding partner (e.g. a peptide).The affinity of a molecule for its binding partner can generally berepresented by the dissociation constant (KD). Affinity can be measuredby common methods known in the art, including those described herein,such as surface plasmon resonance methods, in particular BIAcore.

However, the ability to hold the complex together is not just aboutaffinity. Whilst not wishing to be bound by theory, we hypothesise thatin fact there are three significant components: the on-rate, off-rateand the affinity. The calculation for affinity is based on on-rate andoff-rate. So if the on-rate is low and the off-rate is fast, then theaffinity will be low and that will not be sufficient to hold thebispecific protein complex together. However, a slow on-rate could becompensated for by a slow off-rate giving an overall suitable affinity.In some embodiments a high on-rate may be sufficient to hold the complextogether.

If the binding partners (X and Y) employed in the complex have a slowon-rate then additional time may be required after mixing the componentsto allow the complex to form.

If the affinity between the binding partners is sufficiently high, itmay be possible for the bispecific protein complex to perform itsdesired biological function even if the affinity of the proteins (A andB) of the bispecific protein complex only bind weakly to their targets.Conversely, if the proteins (A and B) are able to bind strongly to theirtargets, it may be possible to achieve the same biological function evenif the affinity of the binding partners (X and Y) for each other islower. In other words, a ‘trinity’ relationship exists such that ahigher affinity between the binding partners can compensate for a loweraffinity for the targets and vice versa.

In one embodiment the affinity of protein A for its ligand or antigen isabout 100 nM or stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500pM, 250 pM, 200 pM, 100 pM or stronger, in particular a binding affinityof 50 pM or stronger.

In one embodiment the affinity of protein B for its ligand or antigen isabout 100 nM or stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500pM, 250 pM, 200 pM, 100 pM or stronger, in particular a binding affinityof 50 pM or stronger.

In one embodiment an interaction between a constant domain in a heavychain, such as CH1 and a constant domain in a light chain, such asCKappa contribute to the formation and/or stability of a bispecificcomplex according to the present disclosure. Thus employing Fab or Fab′fragments in the bispecific complexes of the present disclosure isbeneficial.

In one embodiment the bispecific complex of the present disclosure doesnot comprise a component with an effector function, for example thecomplex does not comprise a constant domain other than a CH1 and CKappaor CLambda, in particular does not comprise constant domainsindependently selected from the group comprising CH2, CH3, CH4 andcombinations thereof. In one embodiment the bispecific complex of thepresent disclosure lacks an Fc region.

In one embodiment the method herein is employed to screen a naïve phagelibrary by preparing fusion proteins of the disclosure from the library.

The bispecific protein complexes of the present invention may be used inany suitable application, including functional screening. This novelformat is particularly useful in multiplex functional screening toidentify protein targets based on function, and optimal epitopes onthose target proteins, which could be targeted by bispecific therapies.Furthermore where proteins A and B are antibodies or binding fragmentsthereof the bispecific protein complexes may also be used for multiplexfunctional screening to identify optimal variable region pairs for usein bispecific antibody therapeutics.

“Multiplex” as employed herein is a population of entities for testingcomprising:

-   -   at least two component fusion proteins (A-X and Y-B) combined to        generate at least one heterodimerically-tethered bispecific        protein complex and at least one relevant biological comparator        in the same or a different format, or    -   at least two heterodimerically-tethered bispecific protein        complexes with optionally at least one relevant biological        comparator in the same or a different format.

Clearly to be useful, the different format employed as the comparatormust be suitable for testing in a functional in vitro assay employed inthe disclosure. In one example the comparator in the multiplex is amonovalent mixture of A-X and B-X or a bivalent monospecific complex ofA-X-Y-A.

In one embodiment the multiplex comprises 1 to hundreds of thousands ofheterodimerically-tethered bispecific protein complexes, for example 2to 500,000 of said complexes, such as 2 to 100,000 or 2 to 10,000, inparticular generated from mixing in a grid 2 to 100 s of first andsecond fusion proteins (A-X and B-Y). In one embodiment the multiplexcomprises for example 2 to 1,000, such as 2 to 900, 2 to 800, 2 to 700,2 to 600, 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 2 to 90, 3to 80, 4 to 70, 5 to 60, 6 to 50, 7 to 40, 8 to 30, 9 to 25, 10 to 20 or15 bispecific protein complexes. See FIG. 3 for an example of such agrid.

In one embodiment the number of heterodimerically-tethered bispecificproteins in this multiplex is n² where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more.

The multiplex may be in the form of an array, for example a microtitreplate, wherein each well of the microplate may contain a differentbispecific protein complex. The bispecific protein complexes may betethered to a solid substrate surface, for example attached to a bead,or they may be suspended in a liquid (e.g. a solution or media) form,for example within a well or within a droplet.

In one embodiment every ‘A’ in the multiplex is a different protein,preferably an antibody or binding fragment thereof that binds to atarget antigen and every ‘B’ is a different protein preferably anantibody or binding fragment thereof that binds to a target antigen.

In one embodiment the multiplex is provided in a grid as discussedbelow, for example an 8×8, 16×16 or 16×20, which equates to 64, 256 or320 samples respectively.

“Grid” as employed herein refers to a two dimensional plot or arraywhere one variable, such a protein A (in A-X) is varied along one axis,such as the X-axis (horizontal axis) and another variable such asprotein B (in B-Y) is varied along the other axis, such as the Y axis(vertical axis). This arrangement assists in systematically evaluatingthe various combinations (permutations) of the variables.

In one embodiment the multiplex is provided on 96 well plates and thesamples analysed may be multiples thereof i.e. 96, 192, 384 etc.

Advantageously, a grid arrangement is particularly advantageous forefficiently screening the biological function of bispecific proteincomplexes according to the present disclosure.

FIG. 3 shows an example of such a grid, whereby 4 first fusion proteinscan be readily combined with 4 second fusion proteins to produce 16bispecific protein complexes.

Other variations of a screening grid will be apparent to the skilledaddressee, for example the first protein (A) in the first fusion protein(A-X) may be kept constant whilst the second protein (B) in the secondfusion protein (B-X) is varied. This may be useful for quickly screeninga large number of different second proteins for synergistic functionwith the pre-selected first protein.

In another embodiment, protein A is varied along one axis by changingthe antibody variable regions of protein A such that each antibodyvariant is specific for the same antigen but has a different combinationof variable regions. Protein B may either be kept constant or may alsobe varied in the same fashion or varied such that the antigenspecificity changes (across or down the grid) for the B proteins.

Advantageously, such a screening grid may potentially allow for thedetection of small differences in synergistic function when thebispecific protein complexes are specific for the same antigens but withdifferent combinations of variable regions.

In one embodiment, a “common” first fusion protein (A-X) according tothe present disclosure may be present within each well. A range ofdifferent second fusion proteins (B-Y) according to the presentdisclosure may then be dispensed into each well. Subsequently, thespecific binding interaction of the two binding partners (X and Y)physically brings the two fusion proteins together to form thebispecific protein complexes. This results in a multiplex comprisingbispecific protein complexes which all bind to a common first targetantigen (bound by A) but are also capable of binding to a second targetantigen (bound by B) which may be different for each bispecific proteincomplex.

In one embodiment the B-Y fusion proteins comprise different variableregions to the same target antigen to allow optimisation of the variableregions and/or epitopes of the given target antigen bound by B whencombined with the variable regions in A-X.

“Common” first fusion protein as employed herein refers to fusionsproteins wherein the A or B component thereof, bind the same proteins orepitope, in particular where the A or B component have complete identityin the common fusion protein i.e. the common first fusion protein alwayscomprises the same variable region sequence.

The skilled person is also aware of different variations of the above,such that the desired specificities of the bispecific protein complexesat each position in the multiplex can be readily controlled. This allowsfor the efficient screening of different combinations of bispecificprotein complexes when such multiplexes are used in functional assays.In one embodiment factorial design is employed to define the variablesemployed in the grid.

In one embodiment the method of the present disclosure is conducive tohigh-throughput analysis.

In one embodiment, multiple bispecific protein complexes are tested inparallel or essentially simultaneously.

Simultaneously as employed herein refers to the where thesamples/molecules/complexes are analysed in the same analysis, forexample in the same “run”. This may be advantageous as generally thereagents employed for a given sample run will be the same batch,concentration, cell source etc and therefore have the same properties.Furthermore the environmental conditions under which the analysis isperformed, such as temperature and humidity are likely to be similar.

In one embodiment simultaneously refers to concomitant analysis wherethe signal output is analysed by an instrument at essentially the sametime. This signal may require deconvolution to interpret the resultsobtained.

Advantageously, testing multiple bispecific protein complexes allows formore efficient screening of a large number of bispecific proteincomplexes and the identification of new and interesting relationships.

In one embodiment, the multiple bispecific protein complexes are testedby using a multiplex as defined above and subjecting the same to one ormore functional assays. Accordingly the present invention provides amethod for detecting synergistic biological function in aheterodimerically-tethered bispecific protein complex of formula A-X:Y-B

-   -   wherein X:Y is a heterodimeric-tether    -   : is a binding interaction between X and Y,    -   A and B are protein components of the bispecific in the form of        fusion proteins with X and Y respectively, said method        comprising the steps of:        -   (i) testing for activity in a functional assay for part or            all of a multiplex comprising at least one            heterodimerically-tethered bispecific protein complex; and        -   (ii) analysing the readout(s) from the functional assay to            identify or detect synergistic biological function in the            heterodimerically-tethered bispecific protein complex; and    -   wherein Y is an antigen and X is an antibody or binding fragment        thereof specific to Y or X is an antigen and Y is an antibody or        binding fragment thereof specific to X.

The term “biological function” as used herein refers to an activity thatis natural to or the purpose of, the biological entity being tested, forexample a natural activity of a cell, protein or similar. Ideally thepresence of the biological function can be tested using an in vitrofunctional assay, including assays employing mammalian cells, such asliving cells, such as B or T cells, or tissue ex vivo. Natural functionas employed herein also includes aberrant function, such as functionsassociated with diseases, such as cancers.

A relevant “biological comparator” as employed herein refers to asuitable entity for assessing activity, in the same assay as thatemployed for the bispecific protein complex, to establish if there isany change or novel activity or function. Suitable comparators forA-X:Y-B may include a purified protein (including recombinant proteins)in a natural form or presented in the same format as the bispecifice.g., where A and B are the same entity, such as A-X:Y-A or B-X:Y-B i.e.a bivalent monospecific complex. Alternatively the fusion protein A-X orB-Y in an uncomplexed form may be employed as a comparator alone or asan uncomplexed mixture such as A-X and B-X together or A-Y and B-Ytogether. Alternatively, multiple comparators of different formats (inparticular as described herein) may be employed. The person skilled inthe art is able to identify and include a suitable control/comparatorbased on common general knowledge or information that is found in theliterature.

The term “synergistic function” or “synergistic biological function” asused herein refers to a biological activity or level of biologicalactivity or an effect on a biological function or activity that:

-   -   is not observed with individual fusion protein components until        a bispecific is employed (and may include activity observed with        a combination of antibodies to the said antigens, which are not        in an bispecific format, but in particular refers to activity        only observed when the two binding domains are linked in a        bispecific format) or    -   higher or lower activity in comparison to the activity observed        when the first and second proteins of a bispecific protein        complex of the present disclosure are employed individually, for        example activity which is only observed in a bispecific form.

Therefore, “synergistic” includes novel biological function or novelactivity. Synergistic function as employed herein does not generallyinclude simple targeting i.e. based only on binding but will generallyinvolve some inhibition, activation, signalling or similar afterbinding.

Novel biological function or novel activity as employed herein refers toa biological function or activity which is not apparent or is absentuntil the two or more synergistic entities (protein A and protein B) arebrought together (as a bispecific or otherwise) or a previouslyunidentified function.

Higher as employed herein refers to an increase in activity including anincrease from zero e.g. some activity in the bispecific where theindividual uncomplexed bispecific component or components has/have noactivity in the relevant functional assay, also referred to herein asnew activity or novel biological function. Higher as employed hereinalso includes a greater than additive function in the bispecific in arelevant functional assay in comparison to the individual uncomplexedbispecific components (tested alone or in combination with beinglinked), for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300%or more increase in a relevant activity.

In one embodiment the uncomplexed proteins together have the sameactivity as the bispecific and this activity or function was previouslyunknown. This is also a novel synergistic function in the context of thepresent specification.

In one embodiment the synergistic function is a higher function.

In one embodiment the synergistic function is a lower function.

Lower function as employed herein refers to where the bispecific in therelevant functional assay has less or no activity in comparison to theindividual uncomplexed bispecific component (s) which has/have activityin the relevant functional assay, also referred to herein as newactivity or novel biological function (such as a natural protein i.e. arecombinant isolated protein which is not in a fusion protein nor partof any other complex other than one in which occurs in vivo-including anactive domain or fragment of said protein) analysed as an individualprotein or analysed as a mixture of proteins under the same conditions,for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or moredecrease in a relevant activity. Greater than 100% decrease in activityrefers to a gain in positive activity in a different direction, forexample where an entity is an agonist decrease in activity over 100% mayrender the entity an antagonist and vice versa.

In one embodiment the activity of the bispecific complex is lower thanthe sum of the known function of protein A and protein B.

In some embodiments the bispecific protein complexes of the presentdisclosure have simply additive biological function. Additive biologicalfunction as employed herein refers to function, which is the same as thesum of each of the components A and B individually, when tested underthe same conditions. An additive function may be a novel function if theactivity or function was previously unknown or unidentified.

Screening is performed using any suitable assay known in the art,depending on the desired function to be identified.

In one embodiment, the functional assay employed in a method of thepresent disclosure is an in vitro or ex vivo assay.

A “functional assay,” as used herein, is an assay that can be used todetermine one or more desired properties or activities of the bispecificprotein complexes, antibody complexes or the mixture of antibodiessubject to the assay conditions. Suitable functional assays may bebinding assays, apoptosis assays, antibody-dependent cellularcytotoxicity (ADCC) assays, complement-dependent cytotoxicity (CDC)assays, inhibition of cell growth or proliferation (cytostatic effect)assays, cell-killing (cytotoxic effect) assays, cell-signalling assays,cytokine production assays, antibody production and isotype switching,cellular differentiation assays, colony forming assays, chemotaxisassays, cell adhesion assays, cell migration assays, cell cycle assays,metabolic assays (whole cell and organelle function), assays formeasuring inhibition of binding of pathogen to target cell, assays tomeasure the secretion of vascular endothelial growth factor (VEGF) orother secreted molecules, assays for bacteriostasis, bactericidalactivity, neutralization of viruses, assays to measure the attraction ofcomponents of the immune system to the site where antibodies are bound,including in situ hybridization methods, labeling methods, and the like.

In one embodiment in vivo assays, such as animal models, including mousetumor models, models of auto-immune disease, virus-infected orbacteria-infected rodent or primate models, and the like, may beemployed.

The skilled person is well able to select a suitable functional assaybased on the target/proteins being investigated. However, the complexesmay be subject to a panel of “standard” assays without preselectingassays thought to be relevant in an attempt identify new functionality.

In the context of bispecific antibody complexes, the efficacy ofbispecific antibody complexes according to the present disclosure can becompared to individual antibodies or mixtures of antibodies (orfragments) in such models by methods generally known to one of ordinaryskill in the art.

For example, the bispecific antibody complexes may be tested for theability to inhibit proliferation, affect viability or metabolic activityof cells (for example with a stain such as allamar blue or by monitoringluminescence due to luciferase expressed by the cells), or causeapoptosis of cancer cells, which are biological functions that includeproperties other than binding to an antigen.

By choosing functional assays closely related to a particular disease ofinterest, the methods of the disclosure make it possible to identifypotentially therapeutic antibodies that bind to known or unknown targetmolecules. It is thus possible to identify new target molecules and/orto directly identify potentially therapeutic antibodies using themethods of the disclosure. Advantageously, the present method is notlimited to any particular assay(s) and provides the user with completeflexibility to select the most appropriate functional assay depending onthe requirements.

When screening the bispecific antibody complexes for desired biologicalfunction, various strategies may be employed. For example, mediumcontaining the antibodies can be directly screened for the biologicalactivity. Alternatively, the antibodies can be bound to beads coated orto microtiter plates prior to screening for biological activity.Alternatively a fusion protein maybe purified via a His tag in a nickelcapture purification step. Such strategies may increase localconcentrations of the antibodies leading to clearer results from thefunctional assays.

The functional assays may be repeated a number of times as necessarywith or without different samples of a particular bispecific antibodycomplex to enhance the reliability of the results. Various statisticaltests known to the skilled person can be employed to identifystatistically significant results and thus identify bispecific antibodycomplexes with biological functions.

When establishing a functional assay for screening the skilled personcan set a suitable threshold over which an identified activity is deemeda ‘hit’. Where more than one functional assay is used the threshold foreach assay may be set at a suitable level to establish a manageable hitrate. In one example the hit rate may be 3-5%. In one example thecriteria set when searching for pairs of antigens that inhibit B cellfunction may be at least 30% inhibition of at least two phospho-readoutsin a B cell activation assay.

In the bispecific protein complexes of the present invention thefollowing protein and peptide components may be used.

In one embodiment, at least one of the first binding partner, X, and thesecond binding partner, Y, of the binding pair are independentlyselected from a peptide and a protein; for example the first bindingpartner or second binding partner is a peptide.

Suitable peptides include the group comprising GCN4, Fos/Jun (human andmurine Fos have a Uniprot number P01100 and P01101 respectively andhuman and murine jun have a Uniprot number 05412 and 05627respectively), HA-tag which correspond to amino acids 98 to 106 of humaninfluenza hemagglutinin, polyhistidine (His), c-myc and FLAG. Otherpeptides are also contemplated as suitable for use in the presentdisclosure and particularly suitable peptides are affinity tags forprotein purification because such peptides have a tendency to bind withhigh affinity to their respective binding partners.

In one embodiment the peptide is not E5B9.

The term “peptide” as used herein refers to a short polymer of aminoacids linked by peptide bonds, wherein the peptide contains in the rangeof 2 to 100 amino acids, for example 5 to 99, such as 6 to 98, 7 to 97,8 to 96 or 5 to 25. In one embodiment a peptide employed in the presentdisclosure is an amino acid sequence of 50 amino acid residues or less,for example 40, 30, 20, 10 or less. The peptides used in the presentdisclosure are of a sufficient length to be fit for purpose, for exampleif the peptide is a linker, it needs to be suitably long to allow thefragment which it links to perform its biological function;alternatively if the peptide is a binding partner, it must be capable ofbinding specifically to another entity such as an antibody.

In one embodiment, the other binding partner of the binding pair (thealternative first or second binding partner) is a protein.

Protein as employed herein refers to an amino acid sequence of 100 aminoacids or more. In one embodiment a “protein” as employed herein refersto an amino acid sequence with a secondary or tertiary structure.

Polypeptide and protein are employed interchangeably herein. However,polypeptide will generally be a protein with a simple structure, forexample little secondary and/or tertiary structure.

In one embodiment the distinction between a peptide and a protein isbased on the presence or absence of secondary structure and/or tertiarystructure, where a peptide has no secondary structure and amino acidswith secondary structure and/or tertiary structure are considered aprotein.

In one embodiment, the protein is an antibody or an antibody fragment.

The term “antibody” as used herein refers to an immunoglobulin moleculecapable of specific binding to a target antigen, such as a carbohydrate,polynucleotide, lipid, polypeptide, peptide etc., via at least oneantigen recognition site (also referred to as a binding site herein),located in the variable region of the immunoglobulin molecule.

As used herein “antibody molecule” includes antibodies and bindingfragments thereof.

“Antibody fragments” as employed herein refer to antibody bindingfragments including but not limited to Fab, modified Fab, Fab′, modifiedFab′, F(ab′)2, Fv, single domain antibodies, scFv, bi, tri ortetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodiesand epitope-binding fragments of any of the above (see for exampleHolliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair andLawson, 2005, Drug Design Reviews—Online 2(3), 209-217). The methods forcreating and manufacturing these antibody fragments are well known inthe art (see for example Verma et al., 1998, Journal of ImmunologicalMethods, 216:165-181). Other antibody fragments for use in the presentdisclosure include the Fab and Fab′ fragments described in Internationalpatent applications WO05/003169, WO05/003170 and WO05/003171.Multi-valent antibodies may comprise multiple specificities e.g.bispecific or may be monospecific (see for example WO92/22853,WO05/113605, WO2009/040562 and WO2010/035012).

A “binding fragment” as employed herein refers to a fragment capable ofbinding a target peptide or antigen with sufficient affinity tocharacterise the fragment as specific for the peptide or antigen.

The term “Fab fragment” as used herein refers to an antibody fragmentcomprising a light chain fragment comprising a VL (variable light)domain and a constant domain of a light chain (CL), and a VH (variableheavy) domain and a first constant domain (CH1) of a heavy chain. In oneexample the heavy chain sequences of the Fab fragment “terminates” atthe interchain cysteine of CH1. In one embodiment the Fab fragmentemployed in a fusion protein of the present disclosure, such as A-Xand/or B-Y is monovalent.

A Fab′ fragment as employed herein refers to a Fab fragment furthercomprising all or part of a hinge region. In one embodiment the Fab′fragment employed in a fusion protein of the present disclosure, such asA-X and/or B-Y is monovalent.

The term “single-chain Fv” or abbreviated as “scFv”, as used hereinrefers to an antibody fragment that comprises VH and VL antibody domainslinked (for example by a peptide linker) to form a single polypeptidechain. The constant regions of the heavy and light chain are omitted inthis format. Single-chain Fv as employed herein includes disulfidestabilised versions thereof wherein in addition to the peptide linker adisulfide bond is present between the variable regions.

Disulfide stabilised scFv may eliminate the propensity of some variableregions to dynamically breath, which relates to variable regionsseparating and coming together again.

The term “single domain antibody” as used herein refers to an antibodyfragment consisting of a single monomeric variable antibody domain.Examples of single domain antibodies include VH or VL or VHH.

In one embodiment the antibody binding fragment and/or the bispecificantibody complex does not comprise an Fc region. “Does not comprise anFc region” as employed herein refers to the lower constant domains, suchas CH2, CH3 and CH4 which are absent. However, constant domains such asCH1, CKappa/CLambda may be present.

In one embodiment, the antibody heavy chain comprises a CH1 domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

In one embodiment, the antibody heavy chain comprises a CH1 domain, aCH2 domain and a CH₃ domain and the antibody light chain comprises a CLdomain, either kappa or lambda.

In one embodiment, the first protein, A, and/or second protein, B, ofthe bispecific protein complex is an antibody or antibody fragment. Sucha bispecific protein complex may be referred to as a bispecific antibodycomplex.

“Bispecific antibody complex” as employed herein refers to a bispecificprotein complex comprising at least two antibody binding sites whereinthe component antibodies, fragments or both are complexed together by aheterodimeric-tether.

A bispecific antibody complex usually refers to a molecule comprising atleast two antigen binding sites, wherein the binding sites havenon-identical specificity.

In one embodiment, the two proteins (for example antibodies, fragmentsor a combination of an antibody and a fragment) target the same antigen,for example binding to two different epitopes on the same targetantigen, also referred to herein as a biparatopic bispecific.

In another embodiment, the two proteins (for example antibodies,fragments or a combination of an antibody and a fragment) may havedifferent antigen specificities, for example binding to two differenttarget antigens.

In yet another embodiment, the two proteins are identical, i.e. bindingto the same epitope on the same target antigen and the complex istherefore monospecific.

In one embodiment each antibody or fragment employed in the bispecificantibody complex of the disclosure comprises one binding site i.e. eachbinding site is monovalent for each target antigen.

The full length antibody or antibody fragment employed in the fusionproteins (A-X or B-Y) may be monospecific, monovalent, multivalent orbispecific.

Advantageously, the use of two bispecific antibody or antibody fragmentsallows the bispecific antibody complex of the present disclosure topotentially be specific for up to 4 different antigens (i.e. the complexmay be tetraspecific). This allows avidity type effects to beinvestigated.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is a monospecific antibody or antibodyfragment, in particular a monovalent Fab, Fab′, scFv, Fv, VHH orsimilar.

In one embodiment, the antibody or antibody fragment employed in thesecond fusion protein (B-Y) is a monospecific antibody or antibodyfragment, in particular a monovalent Fab, Fab′, scFv or similar.

“Monospecific” as employed herein refers to the ability to bind only onetarget antigen.

“Monovalent” as employed herein refers to the antibody or antibodyfragment having a single binding site and therefore only binding thetarget antigen only once.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent, that is has two or morebinding domains.

In one embodiment, the antibody or antibody fragment employed in thesecond fusion protein (B-Y) is multivalent, that is has two or morebinding domains.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is monovalent and the antibody or antibodyfragment employed in the second fusion protein (B-X) is monovalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is monovalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is multivalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is monovalent.

In one embodiment, the antibody or antibody fragment employed in thefirst fusion protein (A-X) is multivalent and the antibody or antibodyfragment employed in the second fusion protein (B-Y) is multivalent.

In one embodiment A-X or B-Y is not a fusion protein comprising twoscFvs one specific to the antigen CD33 and one specific to the antigenCD3 or alternatively a bispecific complex format specific to these twoantigens.

In one embodiment the A-X or B-Y is not a fusion protein comprising ascFv (or alternatively another antibody format) specific to CD3 linkedto a peptide E5B9.

A “binding domain or site” as employed herein is the part of theantibody that contacts the antigen/epitope and participates in a bindinginteraction therewith. In one embodiment the binding domain contains atleast one variable domain or a derivative thereof, for example a pair ofvariable domains or derivatives thereof, such as a cognate pair ofvariable domains or a derivative thereof.

In one embodiment the binding domain comprises 3 CDRs, in particularwhere the binding domain is a domain antibody such as a VH, VL or VHH.In one embodiment the binding domain comprises two variable domains and6 CDRs and a framework and together these elements contribute to thespecificity of the binding interaction of the antibody or bindingfragment with the antigen/epitope.

A “cognate pair” as employed herein refers to a heavy and light chainpair isolated from a host as a pre-formed couple. This definition doesnot include variable domains isolated from a library, wherein theoriginal pairings from a host is not retained. Cognate pairs may beadvantageous because they are often affinity matured in the host andtherefore may have high affinity for the antigen to which they arespecific.

A “derivative of a naturally occurring domain” as employed herein isintended to refer to where one, two, three, four or five amino acids ina naturally occurring sequence have been replaced or deleted, forexample to optimize the properties of the domain such as by eliminatingundesirable properties but wherein the characterizing feature(s) of thedomain is/are retained. Examples of modifications are those to removeglycosylation sites, GPI anchors, or solvent exposed lysines. Thesemodifications can be achieved by replacing the relevant amino acidresidues with a conservative amino acid substitution.

In one embodiment, the bispecific antibody complexes of the presentdisclosure or antibody/fragment components thereof are processed toprovide improved affinity for a target antigen or antigens. Suchvariants can be obtained by a number of affinity maturation protocolsincluding mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403,1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783,1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol.,250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin.Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391,288-291, 1998). Vaughan et al. (supra) discusses these methods ofaffinity maturation.

In one embodiment, the first antibody or antibody fragment (A) isspecific to a first antigen and the second antibody or antibody fragment(B) is specific to a second antigen, wherein the first and secondantigens are different. Advantageously, the bispecfic antibody complexmay be specific for two different antigens. This presents thepossibility of the antibody complex binding to two different antigens,each located on a different entity, thereby bringing the two entitiesinto close physical proximity with each other.

Alternatively, the first antibody or antibody fragment (A) may bespecific for a first epitope and the second antibody or antibodyfragment (B) may be specific for a second epitope, wherein the first andsecond epitopes are both on the same antigen. This can greatly enhancethe avidity of the bispecific antibody complex for the antigen due tothe multiple interactions between the antigen and bispecific antibodycomplex.

In one embodiment, the first antibody (A) or second antibody (B) or boththe first and second antibody of a bispecific antibody complex of thepresent disclosure may be an IgG, optionally with an inactive or activeFc region.

In one embodiment, the first (A) or second (B) antibody fragment isselected from the group consisting of: a fragment antigen binding (Fab),a Fab′, a single chain variable fragment (scFv) and a single domainantibody (sdAb), such as a VHH.

In one embodiment, the first antibody/fragment (A), secondantibody/fragment (B) or both the first and second antibody/fragment ofthe bispecific antibody complex of the present disclosure may be a Fab.

In one embodiment, the first antibody/fragment (A), secondantibody/fragment (B) or both the first and second antibody/fragment ofthe bispecific antibody complex of the present disclosure may be a Fab′.

In one embodiment, the first antibody/fragment (A), secondantibody/fragment (B) or both the first and second antibody/fragment ofthe bispecific antibody complex of the present disclosure may be a scFv.

In one embodiment, the first (A) or second (B) antibody/fragment or boththe first and second antibody/fragment of the bispecific antibodycomplex of the present disclosure is/are a VHH.

For convenience bispecific protein complexes of the present disclosureare referred to herein as A-X:Y-B. However, this nomenclature is notintended to limit how the fusion protein A-X and B-Y are designedbecause our experiments indicate that binding partners X and Y can bereversed i.e. A-Y and B-X without adversely impacting on the method.Thus A and B and X and Y are nominal labels referred to for assistingthe explanation of the present technology.

“Attached” as employed herein refers to connected or joined directly orindirectly via a linker, such as a peptide linker examples of which arediscussed below. Directly connected includes fused together (for examplea peptide bond) or conjugated chemically.

“Binding partner” as employed herein refers to one component part of abinding pair.

In one embodiment, the affinity of the binding partners is high, 5 nM orstronger, such as 900, 800, 700, 600, 500, 400, 300 pM or stronger.

“Binding pair” as employed herein refers to two binding partners whichspecifically bind to each other. Examples of a binding pair include apeptide and an antibody or binding fragment specific thereto, or anenzyme and ligand, or an enzyme and an inhibitor of that enzyme.

In one embodiment, the first binding partner (X) is selected from thegroup comprising: a full length antibody, a Fab, a Fab′, Fv, dsFv, ascFv and a sdAb, wherein examples of a sdAb include VH or VL or VHH.

When X is an antibody or binding fragment thereof then Y is a protein orpeptide, in particular a peptide.

In one embodiment, the second partner (Y) is selected from the groupcomprising: a full length antibody, a Fab, a Fab′, Fv, dsFv, a scFv anda sdAb, wherein examples of a sdAb include VH or VL or VHH.

When Y is an antibody or binding fragment thereof then X is a protein orpeptide, in particular a peptide.

In one embodiment, where A is an antibody or fragment thereof the firstbinding partner (X) is attached to the C-terminal of the heavy or lightchain of the first antibody or antibody fragment, for example, the firstbinding partner (X) is attached to the C-terminal of the heavy chain ofthe first antibody or antibody fragment (A).

In another embodiment, where B is an antibody or fragment thereof thesecond binding partner (Y) is attached to the C-terminal of the heavy orlight chain of the second antibody or antibody fragment, for example thesecond binding partner (Y) is attached to the C-terminal of the heavychain of the second antibody or antibody fragment (B).

In one embodiment X is attached to the C-terminal of the heavy chain ofthe antibody or fragment (protein A) and Y is attached to the C-terminalof the heavy chain of the antibody or fragment (protein B).

In one embodiment X is attached via a linker (such as ASGGGG SEQ ID NO:71 or ASGGGGSG SEQ ID NO: 72) or any other suitable linker known in theart or described herein below, to the C-terminal of the heavy chain ofthe antibody or fragment (protein A) and Y is attached via a linker(such as ASGGGG SEQ ID NO: 71 or ASGGGGSG SEQ ID NO: 72) to theC-terminal of the heavy chain of the antibody or fragment (protein B).

Examples of a suitable binding pair (X or Y) may include GCN4 (SEQ IDNO: 1 or lacking the HIS tag, amino acids 1-38 of SEQ ID NO: 1) or avariant thereof and 52SR4 (SEQ ID NO: 3 or lacking the HIS tag aminoacids 1 to 243 of SEQ ID NO:3) or a variant thereof, which is a scFvspecific for GCN4.

In a one embodiment, the first binding partner (nominally X) is GCN4(for example as shown in SEQ ID NO: 1) or a fragment or variant thereof(for example without the His tag) and the second binding partner(nominally Y) is a scFv or VHH specific for GCN4 (for example as shownin SEQ ID NO: 3) or a variant thereof.

In one embodiment, the first binding partner (nominally X) is a sFv orVHH specific for GCN4 (for example as shown in SEQ ID NO: 3) or avariant thereof and the second binding partner (nominally Y) is GCN4(for example as shown in SEQ ID NO: 1) or a fragment or variant thereof.

GCN4 variants include an amino acid sequence with at least 80%, 85%,90%, 91%, 92%, 93%, 94% 95%, 96%, 97% or 98%, or 99% identity to SEQ IDNO: 1. GCN4 variants also include an amino acid having at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a sequenceencoded by a nucleotide sequence SEQ ID NO: 2, or a sequence encoded bya nucleotide sequence which hybridises to SEQ ID NO: 2 under stringentconditions.

A suitable scFv specific to GCN4 is 52SR4 (SEQ ID NO: 3) or a variantthereof. Variants of 52SR4 include an amino acid sequence with at least80%, or 85%, or 90%, or 95%, or 98%, or 99% identity to SEQ ID NO: 3.52SR4 variants also include an amino acid sequence having at least atleast 80%, or 85%, or 90%, or 95%, or 98%, or 99% to a sequence encodedby a nucleotide sequence SEQ ID NO: 4, or a sequence encoded by anucleotide sequence which hybridises to SEQ ID NO: 4 under stringentconditions.

The present inventors have found that the single chain antibody 52SR4and peptide GCN4, are a binding pair suitable for use in the bispecificprotein complexes of the present disclosure.

Alternatively, any suitable antibody/fragment and antigen (such as apeptide) may be employed as X and Y. Preferably such an X and Y pairresult in greater than 75% heterodimer when A-X and Y-B are combined ina 1:1 molar ratio.

In one embodiment, the first binding partner (X) and the second bindingpartner (Y) are a protein.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is a ligandor vice versa.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is aninhibitor of that enzyme or vice versa.

“Active fragment” as employed herein refers to an amino acid fragment,which is less than the whole amino acid sequence for the entity andretains essentially the same biological activity or a relevantbiological activity, for example greater than 50% activity such as 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment, the first binding partner X is glutathione (GSH)and the second binding partner Y is glutathione-S-transferase (GST) orvice versa.

In another embodiment, X is Fos and Y is Jun or vice versa.

In another embodiment, X is His and Y is anti-His or vice versa.

In another embodiment, the binding pair is clamodulin binding peptideand Y is calmodulin or vice versa.

In another embodiment, X is maltose-binding protein and Y is ananti-maltose binding protein or fragment thereof or vice versa.

Other enzyme-ligand combinations are also contemplated for use inbinding partners. Also suitable are affinity tags known in the art forprotein purification because these have a tendency to bind with highaffinity to their respective binding partners.

“Identity”, as used herein, indicates that at any particular position inthe aligned sequences, the amino acid residue is identical between thesequences. “Similarity”, as used herein, indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. For example, leucine may besubstituted for isoleucine or valine. Other amino acids which can oftenbe substituted for one another include but are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains).

Degrees of identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing. Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST™software available from NCBI (Altschul, S. F. et al., 1990, J. Mol.Biol. 215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J.& Madden, T. L. 1997, Genome Res. 7:649-656,).

In one embodiment, the first or second binding partner (X or Y) is aprotein or peptide.

In one embodiment, the first and second fusion proteins comprise one ormore peptide linkers. The linkers may be incorporated at variouslocations in the fusion proteins. For example, a linker may beintroduced between a binding partner and the protein attached thereto.

In one embodiment, the linker is a peptide linker.

The term “peptide linker” as used herein refers to a peptide with anamino acid sequence. A range of suitable peptide linkers will be knownto the person of skill in the art.

In one embodiment, the binding partners of the bispecific proteincomplexes are joined to their respective proteins via peptide linkers.

In one embodiment the fusion proteins are a translational fusion, thatis a fusion protein expressed in a host cell comprising a geneticconstruct from which the fusion protein is expressed.

In one embodiment the fusion protein is prepared by fusing the heavychain of A to X and/or the heavy chain of B to Y optionally via apeptide linker.

In one embodiment, the peptide linker is 50 amino acids in length orless, for example 20 amino acids or less.

Generally it will be more efficient to express the fusion proteinrecombinantly and therefore a direct peptide bond or a peptide linkerthat can be expressed by a host cell may be advantageous.

In one embodiment, the linker is selected from a sequence shown insequence 5 to 72 or PPP.

TABLE 1 Hinge linker sequences SEQ ID NO: SEQUENCE 5 DKTHTCAA 6DKTHTCPPCPA 7 DKTHTCPPCPATCPPCPA 8 DKTHTCPPCPATCPPCPATCPPCPA 9DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 10 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 11DKTHTCCVECPPCPA 12 DKTHTCPRCPEPKSCDTPPPCPRCPA 13 DKTHTCPSCPA

TABLE 2 Flexible linker sequences SEQ ID NO: SEQUENCE 14 SGGGGSE 15DKTHTS 16 (S)GGGGS 17 (S)GGGGSGGGGS 18 (S)GGGGSGGGGSGGGGS 19(S)GGGGSGGGGSGGGGSGGGGS 20 (S)GGGGSGGGGSGGGGSGGGGSGGGGS 21 AAAGSG-GASAS22 AAAGSG-XGGGS-GASAS 23 AAAGSG-XGGGSXGGGS-GASAS 24AAAGSG-XGGGSXGGGSXGGGS-GASAS 25 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 26AAAGSG-XS-GASAS 27 PGGNRGTTTTRRPATTTGSSPGPTQSHY 28 ATTTGSSPGPT 29 ATTTGS30 GS 31 EPSGPISTINSPPSKESHKSP 32 GTVAAPSVFIFPPSD 33 GGGGIAPSMVGGGGS 34GGGGKVEGAGGGGGS 35 GGGGSMKSHDGGGGS 36 GGGGNLITIVGGGGS 37 GGGGVVPSLPGGGGS38 GGEKSIPGGGGS 39 RPLSYRPPFPFGFPSVRP 40 YPRSIYIRRRHPSPSLTT 41TPSHLSHILPSFGLPTFN 42 RPVSPFTFPRLSNSWLPA 43 SPAAHFPRSIPRPGPIRT 44APGPSAPSHRSLPSRAFG 45 PRNSIHFLHPLLVAPLGA 46 MPSLSGVLQVRYLSPPDL 47SPQYPSPLTLTLPPHPSL 48 NPSLNPPSYLHRAPSRIS 49 LPWRTSLLPSLPLRRRP 50PPLFAKGPVGLLSRSFPP 51 VPPAPVVSLRSAHARPPY 52 LRPTPPRVRSYTCCPTP- 53PNVAHVLPLLTVPWDNLR 54 CNPLLPLCARSPAVRTFP (S) is optional in sequences 17to 20.

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQID NO: 69), PPPP (SEQ ID NO: 70) and PPP.

Other linkers are shown in Table 3:

SEQ ID NO: SEQUENCE 55 DLCLRDWGCLW 56 DICLPRWGCLW 57 MEDICLPRWGCLWGD 58QRLMEDICLPRWGCLWEDDE 59 QGLIGDICLPRWGCLWGRSV 60 QGLIGDICLPRWGCLWGRSVK 61EDICLPRWGCLWEDD 62 RLMEDICLPRWGCLWEDD 63 MEDICLPRWGCLWEDD 64MEDICLPRWGCLWED 65 RLMEDICLARWGCLWEDD 66 EVRSFCTRWPAEKSCKPLRG 67RAPESFVCYWETICFERSEQ 68 EMCYFPGICWM

In one aspect, there is provided a method of producing a bispecificprotein complex of the present disclosure, comprising the steps of:

-   -   (a) producing a first fusion protein (A-X), comprising a first        protein (A), attached to a first binding partner (X) of a        binding pair;    -   (b) producing a second fusion protein (B-Y), comprising a second        protein (B), attached to a second binding partner (Y) of a        binding pair; and    -   (c) mixing the first (A-X) and second fusion proteins (B-Y)        prepared in step a) and b) together.

Typically the mixing of A-X and B-Y in step (c) is in a 1:1 molar ratio.

In one embodiment each fusion proteins employed in the complexes of thepresent disclosure are produced by expression in a host cell or hostcells in an expression experiment.

In one aspect, there is provided a method of preparing a bispecificprotein complex of the present disclosure, comprising the steps of:

-   (a) expressing a first fusion protein (A-X), comprising a first    protein (A), attached to a first binding partner (X) of a binding    pair;-   (b) expressing a second fusion protein (B-Y), comprising a second    protein (B), attached to a second binding partner (Y) of a binding    pair;

wherein fusion protein A-X and B-Y are expressed from the same host cellor distinct host cells.

Distinct host cells as employed herein refers to individual cells,including cells of the same type (even same clonal type).

In one embodiment the expression is transient expression. The use oftransient expression is highly advantageous when combined with theability to generate bispecific complexes without recourse topurification. This results in a rapid method to generate bispecificprotein complexes as transient transfection is much simpler and lessresource intensive than stable transfection.

In one embodiment the expression is stable expression i.e. wherein theDNA encoding the fusion protein in question is stably integrated intothe host cell genome.

In one embodiment a polynucleotide encoding A-X and a polynucleotideencoding B-Y on the same or different polynucleotide sequences aretransfected into a cell as part of a functional assay, wherein theproteins are expressed in the cell and/or released therefrom. Inparticular the polynucleotides are transiently transfected on the sameof different plasmids.

The mixing of A-X and B-Y is generally effected in conditions where theX and Y can interact. In one embodiment, the fusion proteins areincubated in cell culture media under cell culturing conditions, forexample the fusion proteins are incubated for 90 minutes in a 37° C./5%CO₂ environment.

In one embodiment the fusion proteins of the present disclosure aremixed in an aqueous environment, for example one fusion protein may bebound to a solid surface such as a bead or a plate and the other fusionprotein can be introduced thereto in an aqueous solution/suspension. Thesolid phase allows excess components and reagents to be washed awayreadily. In one embodiment neither fusion is attached a solid phase andare simply mixed in a liquid/solution/medium. Thus in one embodiment A-Xand B-Y are mixed as free proteins in an aqueous media.

Advantageously, the method of the present disclosure can be employed toprepare complexes formed between heterogenous pairs (i.e. between thefirst fusion protein [A-X] and second fusion protein [B-Y]) whereininteractions between homogenous pairs (i.e. between two first fusionproteins [A-X] or two second fusion proteins [B-Y]) are minimised. Thusthe present method allows large numbers of bispecific protein complexesto be prepared, with minimal or no contamination with homodimericcomplexes. An advantage of the constructs and method of the presentdisclosure is that the ratio of A-X to B-Y is controlled by theproperties of the A-X and B-Y and in particular a molar ratio of 1:1 canbe achieved. This element of control is a significant improvement overthe certain prior art methods.

In one embodiment a method of the present disclosure comprises a furtherstep of transferring a pair variable regions (in particular two pairs ofvariable regions) identified as having synergistic activity into analternative bispecific format, optionally humanising said variableregions if necessary beforehand, which is an alternative therapeuticformat and/or a format having an extended half-life suitable for testingin assays with a longer duration (for example which run a week or more).

Multivalent formats include those known in the art and those describedherein, such as DVD-Igs, FabFvs for example as disclosed inWO2009/040562 and WO2010/035012, diabodies, triabodies, tetrabodies etc.

Other examples of bi and multispecific formats (including therapeuticformats) include a diabody, triabody, tetrabody, tandem scFv, tandemscFv-Fc, FabFv, Fab′Fv, FabdsFv, Fab-scFv, Fab′-scFv, diFab, diFab′,scdiabody, scdiabody-Fc, ScFv-Fc-scFv, scdiabody-CH₃, IgG-scFv,scFv-IgG, V-IgG, IgG-V, DVD-Ig, and DuoBody.

Diabody as employed herein refers to two Fv pairs: VH/VL and a furtherVH/VL pair which have two inter-Fv linkers, such that the VH of a firstFv is linked to the VL of the second Fv and the VL of the first Fv islinked to the VH of the second Fv.

Triabody as employed herein refers to a format similar to the diabodycomprising three Fv pairs and three inter-Fv linkers.

Tetrabody as employed herein refers to a format similar to the diabodycomprising fours Fv pairs and four inter-Fv linkers.

Tandem scFv as employed herein refers to two scFvs (each comprising alinker is the usual manner) linked to each other via a single linkersuch that there is a single inter-Fv linker.

Tandem scFv-Fc as employed herein refers to two tandem scFvs, whereineach one is appended to the N-terminus of a CH2 domain, for example viaa hinge, of constant region fragment —CH2CH3.

FabFv as employed herein refers to a Fab fragment with a variable regionappended to the C-terminal of each of the following, the CH1 of theheavy chain and CL of the light chain. The format may be provided as aPEGylated version thereof.

Fab′Fv as employed herein is similar to FabFv, wherein the Fab portionis replaced by a Fab′. The format may be provided as a PEGylated versionthereof.

FabdsFv as employed herein refers to a FabFv wherein an intra-Fvdisulfide bond stabilises the appended C-terminal variable regions. Theformat may be provided as a PEGylated version thereof.

Fab-scFv as employed herein is a Fab molecule with a scFv appended onthe C-terminal of the light or heavy chain.

Fab′-scFv as employed herein is a Fab′ molecule with a scFv appended onthe C-terminal of the light or heavy chain.

DiFab as employed herein refers to two Fab molecules linked via theirC-terminus of the heavy chains.

DiFab′ as employed herein refers to two Fab′ molecules linked via one ormore disulfide bonds in the hinge region thereof.

As employed herein scdiabody is a diabody comprising an intra-Fv linker,such that the molecule comprises three linkers and forms a normal scFvwhose VH and VL terminals are each linked to a one of the variableregions of a further Fv pair.

Scdiabody-Fc as employed herein is two scdiabodies, wherein each one isappended to the N-terminus of a CH2 domain, for example via a hinge, ofconstant region fragment —CH2CH3.

ScFv-Fc-scFv as employed herein refers to four scFvs, wherein one ofeach is appended to the N-terminus and the C-terminus of both the heavyand light chain of a —CH2CH3 fragment.

Scdiabody-CH3 as employed herein refers to two scdiabody molecules eachlinked, for example via a hinge to a CH3 domain.

IgG-scFv as employed herein is a full length antibody with a scFv on theC-terminal of each of the heavy chains or each of the light chains.

scFv-IgG as employed herein is a full length antibody with a scFv on theN-terminal of each of the heavy chains or each of the light chains.

V-IgG as employed herein is a full length antibody with a variabledomain on the N-terminal of each of the heavy chains or each of thelight chains.

IgG-V as employed herein is a full length antibody with a variabledomain on the C-terminal of each of the heavy chains or each of thelight chains DVD-Ig (also known as dual V domain IgG) is a full lengthantibody with 4 additional variable domains, one on the N-terminus ofeach heavy and each light chain.

Duobody or ‘Fab-arm exchange’ as employed herein is a bispecific IgGantibody format where matched and complementary engineered amino acidchanges in the constant domains (typically CH3) of two differentmonoclonal antibodies lead, upon mixing, to the formation ofheterodimers. A heavy/light chain pair from the first antibody will, asa result of the residue engineering, prefer to associate with aheavy:light chain pair of a second antibody.

If present constant region domains of a bispecific antibody complex orantibody molecule of the present disclosure, if present, may be selectedhaving regard to the proposed function of the complex or antibodymolecule, and in particular the effector functions which may berequired. For example, the constant region domains may be human IgA,IgD, IgE, IgG or IgM domains. In particular, human IgG constant regiondomains may be used, especially of the IgG1 and IgG3 isotypes when theantibody molecule is intended for therapeutic uses and antibody effectorfunctions are required. Alternatively, IgG2 and IgG4 isotypes may beused when the antibody molecule is intended for therapeutic purposes andantibody effector functions are not required. It will be appreciatedthat sequence variants of these constant region domains may also beused. For example IgG4 molecules in which the serine at position 241 hasbeen changed to proline as described in Angal et al., 1993, MolecularImmunology, 1993, 30:105-108 may be used. Accordingly, in the embodimentwhere the antibody is an IgG4 antibody, the antibody may include themutation S241P.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of posttranslational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995). Accordingly, the C-terminal lysine ofthe antibody heavy chain may be absent.

The present disclosure also provides a composition comprising one ormore bispecific protein complexes as described above, wherein thecomposition predominantly comprises heterodimeric bispecific complexesaccording to the present disclosure, for example with minimal or nocontamination with homodimeric complexes.

In one embodiment, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 90%, or at least 95% of the fusion proteinsin the composition are in a bispecific protein complex form.

In one embodiment, at least 60% of the fusion proteins in thecomposition are in a bispecific protein complex form.

In one embodiment the complexes formed require no further purificationsteps and thus the compositions comprise unpurified bispecificcomplexes.

In one embodiment the complexes formed require one purification step,for example column chromatography.

In one embodiment the method further comprises at least one purificationstep, for example after expression of a fusion protein according to thepresent disclosure and before mixing the fusion proteins.

In one aspect the present disclosure relates to a fusion protein, aheterodimerically-tethered bispecific protein complex, a compositioncomprising a fusion protein or said bispecific protein complex, amultiple, array, library as defined herein.

In one embodiment, the bispecific protein complex is in solution orsuspension.

In one embodiment, the bispecific protein complexes are fixed on a solidsubstrate surface.

In one embodiment, the multiplex is in the form of an array, for examplein a microplate, such as a 96 or 384 well plate. Such arrays can bereadily implemented in screening assays to identify bispecific proteincomplexes with desired functionality.

In another embodiment, the bispecific protein complexes are conjugatedto beads.

A fusion protein as defined above is a component of the bispecificprotein complex according to the present disclosure. In one aspect, thepresent disclosure relates to a fusion protein described herein.

In a further aspect, there is provided a library, comprising two or morefusion proteins as defined above.

The term “library” as used herein refers to two or more bispecificantibody complexes of the present disclosure or multiple fusion proteinsof the present disclosure that can be combined to form at least twodifferent bispecific antibody complexes according to the presentdisclosure. As described throughout the specification, the term“library” is used in its broadest sense and may also encompasssub-libraries.

Advantageously, the library may comprise a range of different fusionproteins which have either the first binding partner (X) or secondbinding partner (Y) of a particular binding pair attached thereto. Inone embodiment part of the library comprisesproteins/antibodies/fragments each connected to a binding partner X andthe remainder of the library comprises the sameproteins/antibodies/fragments each connected to a binding partner Y.This thus allows any two fusion proteins to be readily combined to forma bispecific protein complex of the present disclosure, as long as onefusion protein has the first binding partner of a binding pair attachedand the other fusion protein has the second binding partner of thebinding pair attached.

In one embodiment bispecific protein complexes of the present inventionare suitable for therapeutic applications and may provide noveltherapies for treating diseases. Thus in a further aspect, there isprovided a bispecific protein complex as described above for use intherapy. The bispecific protein complex is suitable for treating a rangeof diseases, such as autoimmune disease and cancer.

Conversely, the bispecific protein complexes of the present disclosurecan be engineered with one antibody or antibody fragment specific forT-lymphocytes, and another antibody or antibody fragment specific for acancer-specific antigen. As a result, the bispecific antibody complexesof the present disclosure may advantageously possess a higher cytotoxicpotential compared to ordinary monoclonal antibodies.

The bispecific protein complexes of the present disclosure are alsoparticularly suited for inhibiting B cell function in order to controlimmune and autoimmune reactions in various autoimmune diseases.

Thus, the present disclosure extends to a method of treating a diseasein a patient, comprising the administration of a bispecific proteincomplex of the present disclosure.

In one aspect, there is provided a pharmaceutical composition comprisingone or more bispecific protein complexes of the present disclosure.

In one embodiment there is provided a fusion protein obtained orobtainable for a method of the present disclosure.

In one embodiment there is provided an bispecific antibody complexobtained or obtainable from a method of the present disclosure

In one embodiment there is provided an a bispecific or multispecificantibody molecule comprising variable regions combinations identified bya method according to the present disclosure.

In one embodiment there is provided a composition, such as apharmaceutical composition comprising a fusion protein, a bispecificantibody complex or a bispecific/multispecific antibody moleculeobtained from a method of the present disclosure.

Various different components can be included in the composition,including pharmaceutically acceptable carriers, excipients and/ordiluents. The composition may, optionally, comprise further moleculescapable of altering the characteristics of the population of antibodiesof the invention thereby, for example, reducing, stabilizing, delaying,modulating and/or activating the function of the antibodies. Thecomposition may be in solid, or liquid form and may inter alia, be inthe form of a powder, a tablet, a solution or an aerosol.

The present disclosure also provides a pharmaceutical or diagnosticcomposition comprising a bispecific protein complex of the presentinvention in combination with one or more of a pharmaceuticallyacceptable excipient, diluent or carrier. Accordingly, provided is theuse of a bispecific protein complex of the invention for use in thetreatment and for the manufacture of a medicament for the treatment of apathological condition or disorder.

The pathological condition or disorder, may, for example be selectedfrom the group consisting of infections (viral, bacterial, fungal andparasitic), endotoxic shock associated with infection, arthritis such asrheumatoid arthritis, asthma such as severe asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, meningoencephalitis,autoimmune uveitis, immune mediated inflammatory disorders of thecentral and peripheral nervous system such as multiple sclerosis, lupus(such as systemic lupus erythematosus) and Guillain-Barr syndrome,Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave'sdisease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere'sdisease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma,Wegener's granulomatosis, other autoimmune disorders, pancreatitis,trauma (surgery), graft-versus-host disease, transplant rejection, heartdisease including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and cancer,including breast cancer, lung cancer, gastric cancer, ovarian cancer,hepatocellular cancer, colon cancer, pancreatic cancer, esophagealcancer, head & neck cancer, kidney, and cancer, in particular renal cellcarcinoma, prostate cancer, liver cancer, melanoma, sarcoma, myeloma,neuroblastoma, placental choriocarcinoma, cervical cancer, and thyroidcancer, and the metastatic forms thereof.

The present disclosure also provides a pharmaceutical or diagnosticcomposition comprising a bispecific protein complex of the presentinvention in combination with one or more of a pharmaceuticallyacceptable excipient, diluent or carrier. Accordingly, provided is theuse of a bispecific protein complex of the invention for use intreatment and in the manufacture of a medicament.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. A pharmaceutical composition of the presentinvention may additionally comprise a pharmaceutically-acceptableadjuvant.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe antibody molecule or bispecific antibody complex of the presentinvention together with one or more of a pharmaceutically acceptableexcipient, diluent or carrier.

The term “pharmaceutically acceptable excipient” as used herein refersto a pharmaceutically acceptable formulation carrier, solution oradditive to enhance the desired characteristics of the compositions ofthe present disclosure. Excipients are well known in the art and includebuffers (e.g., citrate buffer, phosphate buffer, acetate buffer andbicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensionscan be encapsulated in liposomes or biodegradable microspheres. Theformulation will generally be provided in a substantially sterile formemploying sterile manufacture processes.

This may include production and sterilization by filtration of thebuffered solvent solution used for the formulation, aseptic suspensionof the antibody in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragées, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

The bispecific protein complexes of the invention can be delivereddispersed in a solvent, e.g., in the form of a solution or a suspension.It can be suspended in an appropriate physiological solution, e.g.,physiological saline, a pharmacologically acceptable solvent or abuffered solution. Buffered solutions known in the art may contain 0.05mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about4.0 to 5.0. As mentioned supra a suspension can made, for example, fromlyophilised antibody.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

The bispecific antibody complex (or bispecific/multispecific antibodymolecule of the present disclosure) may be the sole active ingredient inthe pharmaceutical or diagnostic composition or may be accompanied byother active ingredients including other antibody ingredients, forexample anti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPSantibodies, or non-antibody ingredients such as xanthines. Othersuitable active ingredients include antibodies capable of inducingtolerance, for example, anti-CD3 or anti-CD4 antibodies.

In a further embodiment, the antibody, fragment or composition accordingto the disclosure is employed in combination with a furtherpharmaceutically active agent, for example a corticosteroid (such asfluticasone propionate) and/or a beta-2-agonist (such as salbutamol,salmeterol or formoterol) or inhibitors of cell growth and proliferation(such as rapamycin, cyclophosphmide, methotrexate) or alternatively aCD28 and/or CD40 inhibitor. In one embodiment the inhibitor is a smallmolecule. In another embodiment the inhibitor is an antibody specific tothe target.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the bispecific antibody complex of the invention (ora bispecific/multispecific antibody molecule of the present disclosure).

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent needed to treat, ameliorate or prevent atargeted disease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose maybe 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg per day.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, theextent of the inflammation present and on whether the antibody moleculeis being used prophylactically or to treat an existing condition. Thefrequency of dose will depend on the half-life of the antibody moleculeand the duration of its effect. If the antibody molecule has a shorthalf-life (e.g. 2 to 10 hours) it may be necessary to give one or moredoses per day. Alternatively, if the antibody molecule has a longhalf-life (e.g. 2 to 15 days) it may only be necessary to give a dosageonce per day, once per week or even once every 1 or 2 months.

In the present disclosure, the pH of the final formulation is notsimilar to the value of the isoelectric point of the antibody orfragment, for if the pH of the formulation is 7 then a pI of from 8-9 orabove may be appropriate. Whilst not wishing to be bound by theory it isthought that this may ultimately provide a final formulation withimproved stability, for example the antibody or fragment remains insolution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a specific tissue ofinterest. Dosage treatment may be a single dose schedule or a multipledose schedule.

Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilising and/or dispersing agents. Alternatively, the bispecificprotein complex (or bispecific/multispecific antibody molecule of thepresent disclosure) may be in dry form, for reconstitution before usewith an appropriate sterile liquid. If the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the bispecific protein complex once it hasbeen absorbed from the gastrointestinal tract.

A nebulisable formulation according to the present disclosure may beprovided, for example, as single dose units (e.g., sealed plasticcontainers or vials) packed in foil envelopes. Each vial contains a unitdose in a volume, e.g., 2 ml, of solvent/solution buffer.

The term “variant” as used herein refers to peptide or protein thatcontains at least one amino acid sequence or nucleotide sequencealteration as compared to the amino acid or nucleotide sequence of thecorresponding wild-type peptide or protein. A variant may comprise atleast 80%, or 85%, or 90%, or 95%, or 98% or 99% sequence identity tothe corresponding wild-type peptide or protein. However, it is possiblefor a variant to comprise less than 80% sequence identity, provided thatthe variant exhibits substantially similar function to its correspondingwild-type peptide or protein.

Antigens include cell surface receptors such as T cell or B cellsignalling receptors, co-stimulatory molecules, checkpoint inhibitors,natural killer cell receptors, Immunolglobulin receptors, TNFR familyreceptors, B7 family receptors, adhesion molecules, integrins,cytokine/chemokine receptors, GPCRs, growth factor receptors, kinasereceptors, tissue-specific antigens, cancer antigens, pathogenrecognition receptors, complement receptors, hormone receptors orsoluble molecules such as cytokines, chemokines, leukotrienes, growthfactors, hormones or enzymes or ion channels, epitopes, fragments andpost translationally modified forms thereof.

In one embodiment, the bispecific protein complex comprises one or twocell surface receptor specificities.

In one embodiment, the bispecific protein complex comprises one or twocytokine or chemokine specificities.

Antibodies or fragments to a pair of targets identified by the methodaccording to the present disclosure may be incorporated into any formatsuitable for use as a laboratory reagent, an assay reagent or atherapeutic.

Thus in one aspect the disclosure extends to use of antibodies fragmentsor combinations thereof as pairs in any format, examples of which aregiven above.

The disclosure also extends to compositions, such as pharmaceuticalcompositions comprising said novel formats with the particular antigenspecificity.

In a further aspect the disclosure includes use of the formats and thecompositions in treatment.

In one embodiment, the bispecific protein complex of the presentdisclosure may be used to functionally alter the activity of the antigenor antigens of interest. For example, the bispecific protein complex mayneutralize, antagonize or agonise the activity of said antigen orantigens, directly or indirectly.

The present disclosure also extends to a kit, for example comprising:

-   a) one or more fusion proteins (A-X) comprising a first antibody or    antibody fragment (A) attached to a first binding partner of a    binding pair (X); and-   b) one or more fusion proteins (B-Y) comprising a second antibody or    antibody fragment (B) attached to a second binding partner of the    binding pair (Y), wherein the latter is specific for the first    binding partner;    -   for example wherein the first binding partner (X) is a peptide        or polypeptide and the second binding (Y) partner is an antibody        or antibody fragment specific thereto;

wherein Y the second binding partner is specific to the first bindingpartner X and the second binding partner is, for example an antibody orantibody fragment specific thereto; and the specific interaction (suchas a binding interaction) of the two binding partners forms aheterodimer-tether which physically brings the two fusion proteins froma) and b) together to form a bispecific protein complex; and

wherein the fusion protein(s) is/are in a complexed or a non-complexedform.

Advantageously, the kit may comprise bispecific protein complexes of thepresent disclosure, or may comprise fusion proteins which are in acomplexed or non-complexed form. In the former case, the bispecificprotein complexes are ready for use “out of the box” which providesconvenience and ease of use, whereas in the latter case, the bispecificprotein complexes can be assembled according to the user's requirementsby combining different fusion proteins.

In another embodiment, the kit further comprises instructions for use.

In yet another embodiment, the kit further comprises one or morereagents for performing one or more functional assays.

In one embodiment, fusion proteins, bispecific proteins complexes,multiplexes, grids, libraries, compositions etc as described herein arefor use as a laboratory reagent.

In a further aspect, there is provided a nucleotide sequence, forexample a DNA sequence encoding a fusion protein and/or a bispecificprotein complex as defined above.

In one embodiment, there is provided a nucleotide sequence, for examplea DNA sequence encoding a bispecific protein complex according to thepresent disclosure.

In one embodiment there is provided a nucleotide sequence, for example aDNA sequence encoding a bispecific or multispecific antibody moleculeaccording to the present disclosure.

The disclosure herein also extends to a vector comprising a nucleotidesequence as defined above.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. An example of a vector is a “plasmid,” which is a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell, where they are subsequently replicated along with the hostgenome. In the present specification, the terms “plasmid” and “vector”may be used interchangeably as a plasmid is the most commonly used formof vector.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

The term “selectable marker” as used herein refers to a protein whoseexpression allows one to identify cells that have been transformed ortransfected with a vector containing the marker gene. A wide range ofselection markers are known in the art. For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. The selectable marker can also be a visuallyidentifiable marker such as a fluorescent marker for example.

Examples of fluorescent markers include rhodamine, FITC, TRITC, AlexaFluors and various conjugates thereof.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present disclosure. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present disclosure. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present disclosure also provides a process for the production of afusion protein according to the present disclosure comprising culturinga host cell containing a vector of the present disclosure underconditions suitable for leading to expression of protein from DNAencoding the molecule of the present disclosure, and isolating themolecule.

The bispecific protein complexes of the present disclosure may be usedin diagnosis/detection kits, wherein bispecific protein complexes withparticular combinations of antigen specificities are used. For example,the kits may comprise bispecific antibody complexes that are specificfor two antigens, both of which are present on the same cell type, andwherein a positive diagnosis can only be made if both antigens aresuccessfully detected. By using the bispecific antibody complexes of thepresent disclosure rather than two separate antibodies or antibodyfragments in a non-complexed form, the specificity of the detection canbe greatly enhanced.

In one embodiment, the bispecific antibody complexes are fixed on asolid surface. The solid surface may for example be a chip, or an ELISAplate.

Further provided is the use of a bispecific protein complex of thepresent disclosure for detecting in a sample the presence of a first anda second peptide, whereby the bispecific complexes are used as detectionagents.

The bispecific antibody complexes of the present disclosure may forexample be conjugated to a fluorescent marker which facilitates thedetection of bound antibody-antigen complexes. Such bispecific antibodycomplexes can be used for immunofluorescence microscopy. Alternatively,the bispecific antibody complexes may also be used for western blottingor ELISA.

In one embodiment, there is provided a process for purifying an antibody(in particular an antibody or fragment according to the invention).

In one embodiment, there is provided a process for purifying a fusionprotein or bispecific protein complex according the present disclosurecomprising the steps: performing anion exchange chromatography innon-binding mode such that the impurities are retained on the column andthe antibody is maintained in the unbound fraction. The step may, forexample be performed at a pH about 6-8.

The process may further comprise an initial capture step employingcation exchange chromatography, performed for example at a pH of about 4to 5.

The process may further comprise of additional chromatography step(s) toensure product and process related impurities are appropriately resolvedfrom the product stream.

The purification process may also comprise of one or moreultra-filtration steps, such as a concentration and diafiltration step.

“Purified form” as used supra is intended to refer to at least 90%purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

In the context of this specification “comprising” is to be interpretedas “including”.

Aspects of the disclosure comprising certain elements are also intendedto extend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements.

Positive embodiments employed herein may serve basis for the excludingcertain aspects of the disclosure.

Disclosures in the context of the method relating to the bispecificcomplexes apply equally to the complexes per se and vice versa.

Paragraphs:

-   1. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex of formula    A-X:Y-B    -   wherein X:Y is a heterodimeric-tether,    -   A and B are components of the bispecific in the form of fusion        proteins with X and Y respectively, said method comprising the        steps of:        -   (i) testing for activity in a functional assay for part or            all of a multiplex comprising at least one            heterodimerically-tethered bispecific protein; and        -   (ii) analysing the readouts from the functional assay to            identify synergistic biological function in the bispecific            protein complex.-   2. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 1, wherein the method further comprises a first step of    forming a multiplex of heterodimerically-tethered bispecific protein    complexes.-   3. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 1 or 2, wherein X is a peptide or protein and Y is a    peptide or protein binding partner specific to X.-   4. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 3, wherein the binding affinity of the    heterodimeric-tether is 5 nM or stronger.-   5. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 4, wherein the binding affinity of the heterodimeric    tether is 900 pM or stronger, such as 800, 700, 600, 500, 400 or 300    pM.-   6. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 3, wherein X is an antibody or binding fragment thereof.-   7. A method for detecting synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 6, wherein the X is an antibody fragment selected from the    group comprising a Fab, a Fab′, a single chain Fv, and a single    domain antibody, such as a VHH.-   8. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 7, wherein the X is a single-chain Fv.-   9. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 8, wherein the single chain Fv is specific to the peptide    GCN4.-   10. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 9, wherein the single chain Fv is 52SR4.-   11. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    any one of paragraph 3 to 10, wherein Y is a peptide.-   12. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    paragraph 11, wherein the peptide is between 5 and 25 amino acids in    length.-   13. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    any one of paragraph 1 to 12, wherein A is an antibody or binding    fragment thereof.-   14. A method for detecting a synergistic function in a    heterodimerically-tethered bispecific protein complex according to    any one of paragraph 1 to 13, wherein B is an antibody or binding    fragment thereof-   15. The method according to any one of the preceding paragraphs    wherein the multiplex comprises at least one biological comparator    for the at least one heterodimerically-tethered bispecific protein.-   16. The method according to any one of paragraph 1 to 15, wherein    multiple bispecific protein complexes are tested simultaneously.-   17. A bispecific protein complex having the formula A-X:Y-B wherein:    -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   A is a first protein component of the bispecific;    -   B is a second protein component of the bispecific;    -   X is a first binding partner of a binding pair;    -   Y is a second binding partner of the binding pair; and    -   : is an interaction (such as a binding interaction) between X        and Y.-   18. The bispecific protein complex according to paragraph 17,    wherein the binding interaction between the X and Y has a low    dissociation constant.-   19. The bispecific protein complex according to paragraph 18 wherein    the dissociation constant is in the range of 1-9×10⁻³ s⁻¹ or less,    for example 1-9×10⁻³ s⁻¹, 1-9×10⁻⁴ s⁻¹, 1-9×10⁻⁵ s⁻¹, 1-9×10⁻⁶ s⁻¹    or 1-9×10⁻⁷ s⁻¹.-   20. The bispecific protein complex according to paragraph 19,    wherein the dissociation constant is 1×10⁻⁴ s⁻¹ or less, for example    1×10⁻⁵ s⁻¹, 1×10⁻⁶ s⁻¹ or 1×10⁻⁷ s⁻¹. 21. The bispecific protein    complex according to any one of paragraphs 17 to 20, wherein the    affinity of X and Y for each other is 5 nM or stronger, for example    900 pM or stronger, such as 800, 700, 600, 500, 400 or 300 pM.-   22. The bispecific protein complex according to any one of    paragraphs 17 to 21, wherein Y is a peptide or protein.-   23. The bispecific protein complex according to paragraph 22,    wherein the peptide is GCN4. 24. The bispecific protein complex    according to any one of paragraph 16 to 23, wherein X is an antibody    or antibody fragment.-   25. A bispecific protein complex according to paragraph 24, wherein    the antibody fragment is selected from the group consisting of: a    Fab, a Fab′, a single chain variable fragment (scFv) and a single    domain antibody (sdAb), such as VHH.-   26. A bispecific protein complex according to paragraph 25, wherein    the antibody fragment is a scFv.-   27. The bispecific protein complex according to paragraph 26,    wherein the scFv is 52SR4.-   28. The bispecific complex according to any one of paragraph 17 to    27, wherein X or Y is a peptide and the peptide is other than the    peptide epitope referred to an E5B9.-   29. The bispecific protein complex according to paragraph 17,    wherein the binding partner pairs are selected from wherein:    -   (i) X is glutathione (GSH) and Y is glutathione S-transferase        (GST),    -   (ii) X is Fos and Y is Jun,    -   (iii) X is FLAG and Y is an anti-FLAG antibody or fragment        thereof,    -   (iv) X is His and Y is anti-His, and    -   (v) X is maltose-binding protein and Y is an anti-maltose        binding protein or fragment thereof.-   30. The bispecific protein complex according to any one of    paragraphs 17 to 29, wherein A is selected from the group consisting    of an antibody, an antibody fragment, a ligand, a receptor, an    inhibitor and an enzyme, such as an antibody or an antibody    fragment.-   31. The bispecific protein complex according to any one of    paragraphs 17 to 30, wherein B is selected from the group consisting    of an antibody, an antibody fragment, a ligand, a receptor, an    inhibitor and an enzyme, such as an antibody or an antibody    fragment.-   32. The bispecific protein complex according to any one of    paragraphs 30 or 31, wherein A and/or B is an antibody or antibody    fragment specific for a cell surface receptor.-   33. The bispecific protein complex according to any one of    paragraphs 29 to 30, wherein A and/or B is an antibody or antibody    fragment is specific for a cytokine or chemokine.-   34. The bispecific protein complex according to any one of    paragraphs 30 to 34 wherein X is attached to the C-terminal of the    heavy or light chain of the first antibody or antibody fragment, for    example wherein X is attached to the C-terminal of the heavy chain    of the first antibody or antibody binding fragment.-   35. The bispecific protein complex according to any one of    paragraphs 31 to 35, wherein Y is attached to the C-terminal of the    heavy or light chain of the second antibody or antibody binding    fragment, for example wherein the Y is attached to the C-terminal of    the heavy chain of the second antibody or antibody fragment.-   36. The bispecific protein complex according to any one of    paragraphs 17 to 36, wherein A is an antibody or antibody binding    fragment specific to a first antigen and B is an antibody or    antibody fragment specific to a second antigen, wherein the first    and second antigens are different.-   37. A composition comprising one or more bispecific protein    complexes according to any one of paragraphs 17 to 37.-   38. The composition according to paragraphs 38, wherein at least    60%, at least 65%, at least 70%, at least 75%, at least 80%, at    least 85%, at least 90%, or at least 95% of the fusion proteins are    in a bispecific protein complex form.-   39. The composition according to paragraphs 39, wherein at least 60%    of the fusion proteins are in a bispecific protein complex form.-   40. A multiplex of bispecific protein complexes, comprising:

at least two bispecific protein complexes according to any one ofparagraphs 19 to 37, wherein the bispecific protein complexes havedifferent specificities.

-   41. The multiplex according to paragraphs 41, wherein the bispecific    protein complexes are in solution or fixed on a solid substrate    surface.-   42. A multiplex according to paragraphs 42, wherein the multiplex is    in the form of an array or a grid, for example in a microplate, such    as a 96 well plate or 384 well plate.-   43. A multiplex according to any one of paragraphs 41 to 43, wherein    the bispecific protein complexes are conjugated to beads.-   44. A fusion protein A-X or B-Y as defined in any one of paragraphs    1 to 37.-   45. A library comprising two or more fusion proteins as defined in    any one of paragraphs 1 to 37.-   46. A nucleotide sequence encoding a fusion protein as defined in    any one of paragraphs 1 to 37.-   47. A vector comprising a nucleotide sequence according to    paragraphs 47.-   48. A bispecific protein complex according to any one of paragraphs    17 to 37 or a composition according to any one of paragraphs 38 to    40, for use in therapy.-   49. A method of treating a patient, comprising the administration of    a bispecific protein complex according to any one of paragraphs 7 to    36 or a composition according to any one of paragraphs 37 to 39.-   50. A kit, comprising:    -   a) one or more fusion proteins A-X; and    -   b) one or more fusion proteins B-Y;        -   X:Y is a heterodimeric-tether;        -   A is a first protein component on of the bispecific;        -   B is a second protein of the bispecific;        -   X is a first binding partner of a binding pair, such as a            peptide or protein;        -   Y is a second binding partner of the binding pair, for            example a peptide or protein specific to X; and    -   wherein X and Y are incapable of forming homodimers, such that a        specific binding interaction between X and Y forms a        heterodimeric-tether and physically brings the two fusions        together to form a bispecific protein complex; and    -   wherein the fusion protein(s) in the kit is/are in a complexed        or a non-complexed form.-   51. The kit according to paragraph 51, further comprising    instructions for use.-   52. The kit according to paragraph 50 or 51, further comprising one    or more reagents for performing one or more functional assays.

All references referred to herein are specifically incorporated byreference.

REFERENCES

-   1. Ribosome display efficiently selects and evolves high-affinity    antibodies in vitro from immune libraries. Hanes J, Jermutus L,    Weber-Bornhauser S, Bosshard H R, Plückthun A. (1998) Proc. Natl.    Acad. Sci. U.S.A. 95, 14130-14135-   2. Directed in Vitro Evolution and Crystallographic Analysis of a    Peptide-binding Single Chain Antibody Fragment (scFv) with Low    Picomolar Affinity. Zhand C, Spinelli S, Luginbuhl B, Amstutz P,    Cambillau C, Pluckthun A. (2004) J. Biol. Chem. 279, 18870-18877-   3. Antigen recognition by conformational selection. Berger C,    Weber-Bornhauser S, Eggenberger Y, Hanes J, Pluckthun A,    Bosshard H. R. (1999) F.E.B.S. Letters 450, 149-153

EXAMPLES

General methods employed in some of the Examples

General Method 1:

Human PBMC derived from platelet apheresis cones were banked as frozenaliquots. Prior to an assay being performed, cells were thawed, washedin DMEM (Life Technologies) and allowed to acclimatise to a 37° C. and5% CO₂ environment.

General Method 2:

Fab′A-X and Fab′B-Y were incubated together for 90 minutes (in a 37°C./5% CO₂ environment) before mixing with 2.5×10⁵ PBMC in V-bottomed 96well plates. PBMC plus bispecific (Fab′A-X and Fab′B-Y) or bivalent(e.g. Fab′A-X FabA′-Y) combinations were then incubated together for afurther 90 minutes. After this time B cells were activated by theaddition of 200 nM of goat F(ab′)2 anti-human IgM (SouthernBiotechnology) for 8 minutes at 37° C. The signalling reaction was thenhalted by adding an equal volume of Cytofix buffer (BD Biosciences).Plates were then left at room temperature for 15 minutes beforecentrifugation at 500 g for 5 minutes. Excess supernatant was discardedfrom the cell pellet which was resuspended in flow buffer and washedonce more. Cells were then resuspended in ice cold Perm Buffer III (BDBiosciences) for 30 minutes before being washed twice in flow buffer.

General Method 3:

Cells were activated as described in general method 2 and stained with afluorescently labelled anti-CD20 antibody (BD Biosciences), anti-phosphoAkt antibody that recognises a modified serine residue at position 473,an anti-phospho PLCg2 antibody that recognises a modified tyrosineresidue at position 759 and an anti-IκB antibody that recognised totalIκB. Plates were then resuspended and incubated for 1 hour at roomtemperature in the dark. After this time plates were washed a furthertwo times and resuspended in 25 μl of flow buffer. Cellular expressionof CD20, Akt and PLCg2 was measured using an Intellicyt HTFC™ flowcytometer

Example 1 Construction of a Bispecific Antibody Complex of the PresentDisclosure FabB-GCN4(7P14P):52SR4-FabA

FIGS. 2 and 4 show a representative bispecific antibody complex of thepresent disclosure. The bispecific antibody complex is composed of afirst and second fusion protein.

The first fusion protein (A-X) includes a Fab fragment (Fab A (alsoreferred to as Fab#1) with specificity to antigen 6, which is attachedto X a scFv (clone 52SR4 SEQ ID NO: 3) via a peptide linker ASGGGG SEQID NO: 71 which is linked to the c-terminal of the CH₁ domain of the Fabfragment and the V_(L) domain of the scFv. The scFv itself also containsa peptide linker located in between its V_(L) and V_(H) domains.

The second fusion protein (B-Y) includes a Fab fragment (Fab B [alsoreferred to as Fab#2] with specificity to antigen 5). However, incomparison to the first protein, the Fab fragment is attached to Y apeptide GCN4 (clone 7P14P SEQ ID NO: 1) via a peptide linker ASGGGG SEQID NO: 71 which is linked to the CH₁ domain of the Fab fragment.

The scFv, X, is specific for and complementary to the binding partner Y,GCN4. As a result, when the two fusion proteins are brought into contactwith each other, a non-covalent binding interaction between the scFv andGCN4 peptide occurs, thereby physically retaining the two fusionproteins in the form of a bispecific antibody complex.

The single chain antibody (scFv) 52SR4 was derived by constructing andpanning a ribosome display VL-linker-VH scFv library from the spleens ofmice immunized with GCN4(7P14P) (Reference 1). A further 2004publication describes the affinity maturation of 52SR4 to a reported 5pM again using ribosome display of randomised libraries (Reference 2).

The GCN4 peptide was derived from the yeast transcription factor GCN4 byinclusion of Proline residues at positions 7 and 14, hence GCN4(7P14P)remains in a monomeric state on scFv binding as described in a 1999publication by Berger et al (Reference 3).

The nucleotide sequences encoding the GCN4 peptide and the 52SR4 scFvwere cloned into two separate vectors downstream of in-house heavy chainFab expression vectors which contain CH₁ and which are already designedto receive antibody VH-regions.

VH-regions from an anti-antigen 6 antibody and an anti-antigen 5antibody were then cloned separately into these two heavy chain vectors.

The nucleotide sequences encoding the GCN4 peptide and the 52SR4 scFvwere separately cloned into a first and second vector respectivelydownstream of in-house light chain Fab expression vectors which containCK and which are designed to receive antibody VL-regions.

VL-regions from an anti-antigen 6 antibody and an anti-antigen 5antibody were cloned separately in frame with CK in an in-house lightchain expression vector for co-expression with the appropriate heavychain vector to express the Fab-scFv and Fab-peptide proteins.

The vectors were then sequenced to confirm that the cloning has beensuccessful and that the cells subsequently separately expressed Fab-scFvand Fab-peptide proteins with the V-regions from the anti-antigen 6antibody and the anti-antigen 5 antibody respectively. Antigen 5 and 6in Example 1 are not the antigens labelled antigen 5 and antigen 6 inlater Examples with the large grid formats.

Example 2—Flow Cytometry Demonstration of scFv:Peptide InteractionForming a Non-Covalent Bispecific Antibody that can Co-Engage Two TargetAntigens Simultaneously

FIG. 5 shows the results of a flow cytometry experiment whichdemonstrates the antigen specificities of two different bispecificantibody complexes formed using the scFv:peptide binding interaction.

The first bispecific antibody complex was constructed using thefollowing two fusion proteins:

-   -   1. Anti-antigen 5 Fab-scFv (52SR4); and    -   2. Anti-antigen 6 Fab-peptide (GCN4)

The second bispecific antibody complex was constructed using thefollowing two fusion proteins:

-   -   1. Anti-antigen 5 Fab-peptide (GCN4); and    -   2. Anti-antigen 6 Fab-scFv (52SR4)

Therefore, the two bispecific antibody complexes had the same Fabfragments and same binding partners (i.e. 52SR4 and GCN4). Thedifference between the two bispecific antibody complexes was in whichFab fragment is attached to which binding partner.

The control mixture which did not form a complex was made from thefollowing fusion proteins:

-   -   1. Anti-antigen 5 Fab:GCN4; and    -   2. Anti-antigen 6 Fab:GCN4

To demonstrate the ability of the bispecific antibody complexes to bindto antigen 5, the complexes were incubated with Jurkat cells whichexpress antigen 5. To demonstrate the ability of the bispecific antibodycomplexes to bind to antigen 6, the complexes once bound to antigen 5 onJurkat cells were subsequently contacted with biotinylated antigen 6.The biotinylated antigen 6 was then detected using fluorescentlylabelled streptavidin.

The Jurkat cells were then run through a Facscalibur flow cytometermachine, wherein the fluorescently labelled Jurkat cells which can onlybe labelled when bound to a bispecific antibody complex, which is inturn bound to antigen 6, thereby indicating that the bispecific antibodycomplex is capable of binding to both antigen 5 and antigen 6 can beseparated from Jurkat cells incubated with two fusion proteins capableof binding to antigen 5 and antigen 6, both fused to peptide whichcannot form a complex.

The FACS plot in FIG. 5 shows significant shifts for both the bispecificantibody complexes (thin and thick line over and above backgroundfilled), thus demonstrating that the bispecific antibody complexes cansuccessfully bind to both target antigens and that the ability to bindto both target antigens is retained regardless of whether a given Fabfragment is connected to a scFv or peptide.

The subsequent capture of either peptide or scFv respectivelyC-terminally fused to the anti-antigen 6 Fab allows further capture ofbiotinylated antigen 6 which is detected in a final layer withfluorescently labelled streptavidin. Accordingly, the results depictedin the FACS plot shows that the bispecific antibody complexes of thepresent disclosure are able to successfully bind two different targetantigens simultaneously.

Antigen 5 and 6 in Example 2 are not the antigens labelled antigen 5 andantigen 6 in later Examples with the large grid formats below.

Example 3—Biacore Demonstration of scFv:Peptide Interaction

FIG. 6 shows a surface plasmon resonance trace which demonstrates theaffinity of the scFv:peptide (i.e. 52SR4:GCN4) interaction. Surfaceplasmon resonance was performed using a Biacore 3000 (GE Healthcare).All experiments were performed at 25° C. Streptavidin (producedin-house) was immobilised on a CMS Sensor Chip (GE Healthcare) via aminecoupling chemistry to a final level of approximately 1750 responseunits. HBS-N buffer (10 mM HEPES pH 7.4, 0.15M NaCl; GE Healthcare) wasused as the running buffer for immobilisation and peptide capture. A 5μl injection of Biotin-GCN4 peptide in HBS-N (10 nM, M.W. 4360) was usedto achieve approximately 6RU of capture on the immobilised streptavidin.The running buffer was switched to HBS-EP+ buffer (10 mM HEPES pH 7.4,0.15M NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20; GE Healthcare) formeasuring anti-GCN4 (52SR4) scFv binding kinetics. Three-fold serialdilutions of Fab-scFv (generated in-house) from 30 nM, or HBS-EP+ buffercontrol, were injected over the immobilised GCN4 peptide (3 minassociation, 15 min dissociation) at a flow rate of 30 μl/min. Thesurface was regenerated after each injection at a flow-rate of 10 μl/minby two serial 60 sec injection of 2M Guanidine-HCl. Double referencedbackground subtracted binding curves were analysed using the 3000BIAEval software (version 4.1) following standard procedures. Kineticparameters were determined from fitting the 1:1 binding model algorithm.The data demonstrate that the scFv has an affinity for the peptide of516 pM.

Example 4—Production of Fab-A (Fab-scFv [A-X]) and Fab-B (Fab-Peptide[B-Y) for Functional Assays

Cloning strategy: Antibody variable region DNA was generated by PCR orgene synthesis flanking restriction enzyme sites DNA sequence. Thesesites were HindIII and XhoI for variable heavy chains and HindIII andBsiWI for variable light chains. This makes the heavy variable regionamenable to ligating into the two heavy chain vectors (pNAFH with FabB-Yand pNAFH with FabA-X) as they have complementary restriction sites.This ligates the variable region upstream (or 5′) to the murine constantregions and peptide Y (GCN4) or scFv X (52SR4) creating a whole readingframe. The light chains were cloned into standard in house murineconstant kappa vectors (pMmCK or pMmCK S171C) which again use the samecomplimentary restriction sites. The pMmCK S171C vector is used if thevariable region is isolated from a rabbit. The cloning events wereconfirmed by sequencing using primers which flank the whole open readingframe.

Cultivating CHOSXE:

Suspension CHOSXE cells were pre-adapted to CDCHO media (Invitrogen)supplemented with 2 mM (100×) glutamx. Cells were maintained inlogarithmic growth phase agitated at 140 rpm on a shaker incubator(Kuner AG, Birsfelden, Switzerland) and cultured at 37° C. supplementedwith 8% CO₂.

Electroporation Transfection:

Prior to transfection, the cell numbers and viability were determinedusing CEDEX cell counter (Innovatis AG. Bielefeld, Germany) and requiredamount of cells (2×10⁸ cells/ml) were transferred into centrifugeconical tubes and were spun at 1400 rpm for 10 minutes. The pelletedcells were re-suspended in sterile Earls Balanced Salts Solution andspun at 1400 rpm for further 10 minutes. Supernatant was discarded andpellets were re-suspended to desired cell density.

Vector DNA at a final concentration of 400 μg for 2×10⁸ cells/ml mix and800 μl was pipetted into cuvettes (Biorad) and electroporated usingin-house electroporation system. Fab-A (Fab-scFv [A-X]) and Fab-B(Fab-peptide [B-Y] were transfected separately. Transfected cells weretransferred directly into 1×3 L Erlenmeyer Flasks contained ProCHO 5media enriched with 2 mM glutamx and antibiotic antimitotic (100×)solution (1 in 500) and cells were cultured in Kuhner shaker incubatorset at 37° C., 5% CO₂ and 140 rpm shaking. Feed supplement 2 g/L ASF(AJINOMOTO) was added at 24 hr post transfection and temperature droppedto 32° C. for further 13 days culture. At day four 3 mM sodium buryrate(n-butric acid sodium Salt, Sigma B-5887) was added to the culture.

On day 14, cultures were transferred to tubes and supernatant separatedfrom the cells after centrifugation for 30 minutes at 4000 rpm. Retainedsupernatants were further filtered through 0.22 um SARTOBRAN® PMillipore followed by 0.22 μm Gamma gold filters. Final expressionlevels were determined by Protein G-HPLC.

Large Scale (1.0 L) Purification:

The Fab-A and Fab-B were purified by affinity capture using the AKTAXpress systems and HisTrap Excel pre-packed nickel columns (GEHealthcare). The culture supernatants were 0.22 μm sterile filtered andpH adjusted to neutral, if necessary, with weak acid or base beforeloading onto the columns. A secondary wash step, containing 15-25 mMimidazole, was used to displace any weakly bound host cellproteins/non-specific His binders from the nickel resin. Elution wasperformed with 10 mM sodium phosphate, pH7.4+1M NaCl+250 mM imidazoleand 2 ml fractions collected. One column volume into the elution thesystem was paused for 10 minutes to tighten the elution peak, andconsequently decrease the total elution volume. The cleanest fractionswere pooled and buffer exchanged into PBS (Sigma), pH7.4 and 0.22 μmfiltered. Final pools were assayed by A280 Scan, SE-HPLC (G3000 method),SDS-PAGE (reduced & non-reduced) and for endotoxin using the PTSEndosafe system.

Example 5-Use of Fab-A (Fab-scFv [A-X]) and Fab-B (Fab-Peptide [B-Y]) inHeterodimerically-Tethered Bispecific Protein Complex Format to SelectFunctional, Bivalent and Bispecific Antigen Target Combinations Based onInhibition of Akt Signalling (as a Measure of B Cell Activation)

Human PBMC were prepared according to general method 1. During thisperiod grids of bispecific or bivalent antibodies were created bydiluting equimolar (200 nM) quantities of Fab′-A (Fab-scFv) and Fab′-B(Fab-peptide) with varying antigen specificity for the cell surfaceproteins antigen 3, antigen 1, antigen 4 and antigen 2 in DMEMcontaining 10% calf serum and 2 mM glutamine. This grid is shown inTable 4.

TABLE 4 Possible grid of bispecific and bivalent combinations ofantibodies with specificity for antigen 3, antigen 1, antigen 4 and 2.(B-Y) Fab B (A-X) Fab A Antigen 3-Y Antigen 1-Y Antigen 4-Y Antigen 2-YAntigen 3-X 3-X:Y-3 3-X:Y-1 3-X:Y-4 3-X:Y-2 Antigen 1-X 1-X:Y-3 1-X:Y-11-X:Y-4 1-X:Y-2 Antigen 4-X 4-X:Y-3 4-X:Y-1 4-X:Y4  4-X:Y-2 Antigen 2-X2-X:Y-3 2-X:Y-1 2-X:Y-4 2-X:Y-2

where X is a scFv (52SR4) and Y is a peptide (GCN4)

Fab′A-X and Fab′B-Y were incubated with PMBCs according to generalmethod 2 following purification as described in Example 4.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences) and a fluorescently labelled anti-phospho Akt antibodythat recognises a modified serine residue at position 473 on theprotein. Plates were then resuspended and incubated for 1 hour at roomtemperature in the dark. After this time plates were washed a furthertwo times and resuspended in 25 μl of flow buffer. Cellular expressionof CD20 and Akt was measured using an Intellicyt HTFC™ flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of Akt levels was calculated for each well. All data wasthen expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). The relative effectof the combinations of antibodies to antigen 3 (VR0982), antibodies toantigen 1 (VR4247), antibodies to antigen 4 (VR4248) and antibodies toantigen 2 (VR4246) is shown in table 5 (↓=inhibition, ↑=stimulation and↔=no overall effect). The number of arrows is indicative of theintensity of the activity.

TABLE 5 Table of the relative potency of inhibition of phosphorylatedAkt for bispecific & bivalent combinations of antibodies withspecificity for antigen 3, 1, 4 & 2 (A-X) Fab A (B-Y) Fab B specific toAntigen 3-Y Antigen 1-Y Antigen 4-Y Antigen 2-Y Antigen 3-X ↑↑ NotTested ↑↑ ↓↓↓ Antigen 1-X ↔ ↔ ↔ ↓↓↓↓ Antigen 4-X Not Tested Not TestedNot Tested Not Tested Antigen 2-X ↓↓↓ ↓↓↓ ↓↓ ↔

where X is a scFv (52SR4) and Y is a peptide (GCN4)

This data is also shown in the form of a histogram (FIG. 7): as meanvalues and the error bars show 95% confidence intervals. The data showsthat the combinations of Fab to antigen 3 (VR0982) with Fab to antigen 2(VR4246), Fab to antigen 1 (VR4247) with Fab to antigen 2 (VR4246) andFab to antigen 4 (VR4248) with Fab to antigen 2 (VR4246) can all inhibitphospho-Akt expression in B cells stimulated with anti-IgM. In contrast,the combinations of Fab to antigen 3 (VR0982) with Fab to antigen 3(VR0982) and Fab to antigen 3 (VR0982) with Fab to antigen 4 (VR4248)exhibited elevated levels of phosho-Akt expression. All othercombinations tested showed no effect.

Example 6-Use of the Heterodimerically-Tethered Bispecific ProteinComplex Format to Select Functional, Bivalent and Bispecific AntigenTarget Combinations Based on Inhibition of PLCg2 Signalling (as aMeasure of B Cell Activation)

Human PBMC were prepared according to general method 1. During thisperiod grids of bispecific or bivalent antibodies were created bydiluting equimolar (200 nM) quantities of Fab′-A (Fab-scFv [A-X]) andFab′-B (Fab-peptide [B-Y]) with antigen specificity for the cell surfaceproteins antigen 3, antigen 1, antigen 4 and antigen 2 in DMEMcontaining 10% calf serum and 2 mM glutamine. This grid is shown inTable 6.

Fab′A-X and Fab′B-Y were incubated according to general method 2.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences) and a fluorescently labelled anti-phospho PLCg2antibody that recognises a modified tyrosine residue at position 759 onthe protein. Plates were then resuspended and incubated for 1 hour atroom temperature in the dark. After this time plates were washed twiceand resuspended in 25 μl of flow buffer. Cellular expression of CD20 andPLCg2 was measured using an Intellicyt HTFC™ flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of PLCg2 levels was calculated for each well. All datawas then expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). The relative effectof the “antibody” combinations to antigen 3, antigen 1, antigen 4 andantigen 2 is shown in table 6 (↓=inhibition, ↑=stimulation and ↔=nooverall effect). The number of arrows is indicative of the intensity ofthe activity.

TABLE 6 Table of the relative potency of inhibition of phosphorylatedPLCg2 for bispecific & bivalent combinations of antibodies withspecificity for antigen 3, 1, 4 & 2. (A-X) Fab A (B-Y) Fab B specific toAntigen 3-Y Antigen 1-Y Antigen 4-Y Antigen 2-Y Antigen 3-X ↑ Not Tested↑↑ ↓↓↓ 1-X ↔ ↔ ↔ ↓↓ 4-X Not Tested Not Tested Not Tested Not Tested 2-X↓↓↓ ↓↓ ↓↓↓ ↔

where X is a scFv and Y is a peptide

This data is also represented as a histogram (FIG. 8), showing meanvalues and the error bars are 95% confidence intervals. The data showsthat the combinations of a Fab to antigen 3 (VR0982) with a Fab toantigen 2 (VR4246), a Fab to antigen 1 (VR4247) with a Fab to antigen 2(VR4246), a Fab to antigen 4 (VR4248) with a Fab to antigen 2 (VR4246)can all inhibit phospho-PLCg2 expression in B cells stimulated withanti-IgM. In contrast, the combinations of a Fab to antigen 3 (VR0982)with a Fab to antigen 3 (VR0982), and a Fab to antigen 3 (VR0982) with aFab to antigen 4 (VR4248) exhibited elevated levels of phosho-PLCg2expression. The combination of a Fab to antigen 1 with a Fab to antigen1 showed no effect.

Example 7—The Use of the Heterodimerically-Tethered Bispecific ProteinComplex Format to Select Functional, Bivalent and Bispecific AntigenTarget Combinations Based on Inhibition of CD86 Expression (as a Measureof B Cell Activation)

Human PBMC were prepared according to general method 1. During thisperiod grids of bispecific or bivalent antibodies were created bydiluting equimolar (200 nM) quantities of Fab′-X (Fab-scFv) and Fab′-Y(Fab-peptide) with antigen specificity for the cell surface proteinsantigen 3, antigen 1, antigen 4 and antigen 2 in DMEM containing 10%calf serum and 2 mM glutamine. This grid is shown in Table 7.

Fab′A-X and Fab′B-Y were incubated together for 90 minutes (in a 37° C.& 5% CO₂ environment) before mixing with 2.5×10⁵ PBMC in V-bottomed 96well plates. PBMC plus bispecific or bivalent combinations were thenincubated together for a further 90 minutes. After this time B cellswere activated by the addition of 200 nM of goat F(ab′)2 anti-human IgM(Southern Biotechnology) for 24 hours at 37° C. After this time plateswere placed on ice and washed once in ice cold flow buffer (PBS+1%BSA+0.01% NaN₃). Cells were then stained with a fluorescently labelledanti-CD19 antibody (BD Biosciences) and a fluorescently labelledanti-CD86 antibody and incubated on ice for 1 hour in the dark. Afterthis time plates were washed a further two times and resuspended in 25μl of flow buffer. Cellular expression of CD19 and CD86 was measuredusing an Intellicyt HTFC™ flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of CD86 levels was calculated for each well. All data wasthen expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). The relative effectof the combinations of Fab to antigen 3 (VR0982), Fab to antigen 1(VR4247), Fab to antigen 4 (VR4248) and Fab to antigen 2 (VR4246) isshown in table 7 (↓=inhibition, ↑=stimulation and ↔=no overall effect).The number of arrows is indicative of the intensity of the activity.

TABLE 7 Table of the relative potency of inhibition of B Cell CD86expression for bispecific and bivalent combinations of antibodies withspecificity for antigen 3, 1, 4 & 2 (A-X) Fab A (B-Y) Fab B specific toAntigen 3-Y Antigen 1-Y Antigen 4-Y Antigen 2-Y Antigen 3-X ↑ Not Tested↔ ↓↓↓ Antigen 1-X ↔ ↔ ↑↑ ↔ Antigen 4-X Not Tested Not Tested Not TestedNot Tested Antigen 2-X ↓↓↓ ↓ ↓↓↓↓ ↓↓where X is a scFv (52SR4) and Y is a peptide (GCN4)

This data is also shown as a histogram (FIG. 9), as mean values and theerror bars are 95% confidence intervals. The data shows that thecombinations of a Fab to antigen 3 (VR0982) with a Fab to antigen 2(VR4246), a Fab to antigen 1 (VR4247) with a Fab to antigen 2 (VR4246),a Fab to antigen 4 (VR4248) with a Fab to antigen 2 (VR4246) and a Fabto antigen 2 (VR4246) with a Fab to antigen 2 (VR4246) can all inhibitCD86 expression on B cells stimulated with anti-IgM. In contrast thecombinations of a Fab to antigen 3 (VR0982) with a Fab to antigen 3(VR0982), and a Fab to antigen 1 (VR4247) with a Fab to antigen 4(VR4248) exhibited elevated levels of CD86 expression. All the othercombinations tested showed no effect.

Example 8—the Inhibitory Effect of a Fab to Antigen 1 (VR4247) and a Fabto Antigen 2 (VR4246) can Only be Reproduced when the Antibodies areArranged in a Bispecific Orientation

Human PBMC were prepared according to general method 1. During thisperiod combinations of bispecific, bivalent or mixtures of antibodieswere created by diluting equimolar (200 nM) quantities of Fab′A-X(Fab-scFv) and/or Fab′B-Y (Fab-peptide) with antigen specificity for thecell surface proteins antigen 1 and antigen 2 in DMEM containing 10%calf serum and 2 mM glutamine. In addition single fab controls (Fab′-Xand Fab′-Y) were also added. These combinations are shown in Table 8.

TABLE 8 Grid of bispecific, bivalent, mixtures or single Fab's withspecificity for antigen 1 and antigen 2 (A-X) Fab A (B-Y) Fab B specificto — Antigen 1-Y Antigen 2-Y Antigen 2-X Antigen 1-X 1-X 1-X:Y-1 1-X:Y-21-X X-2 Antigen 2-X 2-X 2-X:Y-1 2-X:Y-2 — Antigen 1-Y 1-Y — 1-Y Y-2 —Antigen 2-Y 2-Y — — —

where X is a scFv (52SR4) and Y is a peptide (GCN4)

Fab′A-X and/or Fab′B-Y were incubated according to general method 2.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences) and a fluorescently labelled anti-phospho Akt antibodythat recognises a modified serine residue at position 473 on theprotein. Plates were then resuspended and incubated for 1 hour at roomtemperature in the dark. After this time plates were washed a furthertwo times and resuspended in 25 μl of flow buffer. Cellular expressionof CD20 and Akt was measured using an Intellicyt HTFC™ flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of Akt levels was calculated for each well. All data wasthen expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). FIG. 10 shows thatonly the bispecific combination of a Fab to antigen 1 (VR4247) and a Fabto antigen 2 (VR4246) but not any other combination can modulate B cellphospho-Akt levels (the data represents mean values and the error barsare 95% confidence intervals).

Example 9 the Inhibitory Effect of an Anti-Antigen 3 (VR0982) and anAnti-Antigen 2 (VR4246) can Only be Reproduced when the Antibodies areArranged in a Bispecific Orientation

Human PBMC were prepared according to general method 1. During thisperiod combinations of bispecific, bivalent or mixtures of antibodieswere created by diluting equimolar (200 nM) quantities of Fab′-X(Fab-scFv) and/or Fab′-Y (Fab-peptide) with antigen specificity for thecell surface proteins antigen 1 and antigen 2 in DMEM containing 10%calf serum and 2 mM glutamine. These combinations are shown in Table 9.

TABLE 9 Grid of bispecific, bivalent or mixtures with specificity forantigen 3 & 2 (A-X) Fab A (B-Y) Fab B specific to Antigen 3-Y Antigen2-Y Antigen 2-X Antigen 3-X 3-X:Y-3 3-X:Y-2 3-X X-2 Antigen 2-X 2-X:Y-32-X:Y-2 — Antigen 3-Y — 3-Y Y-2 —

where X is a scFv (52SR4) and Y is a peptide (GCN4)

Fab′A-X and/or Fab′B-Y were incubated according to general method 2.

Cells were then stained according to general method 3.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of Akt and PLCg2 levels were calculated for each well.All data was then expressed as the percentage inhibition of the maximalresponse (anti-IgM only) minus the background (cells only). FIGS. 11 and12 show that only the bispecific combination to antigen 3 and antigen 2but not the mixtures of an anti-antigen 3 (VR0982) and an anti-antigen 2(VR4246) antibodies inhibited phosphorylated Akt and PLCg2 expression(the data represents mean values and the error bars are 95% confidenceintervals).

In order to validate the inhibition seen with the bispecific combinationto antigen 3 and to antigen 2, this combination along with a mixture ofanti-antigen 3 (VR0982) and anti-antigen 2 (VR4246) antibodies wastitrated and inhibition of total intracellular IkB (signalling readout)and CD86 (activation marker after 24 hours) was measured in B cells.

As can be seen in FIG. 13, a combination of antigen-3-X/antigen-2-Y butnot the combination of antigen-3-X/antigen-2-X (i.e. as a simpleunlinked mixture) was able to inhibit NF-kB signal activation afteranti-IgM stimulation as measured by the level of total IkB protein. TheIC₅₀, as extrapolated using a 4 parameter logistic curve fit usingGraphpad Prism 6, was 7.5 nM (the data represents mean values and theerror bars are standard deviations). Additionally a titration of thecombination of antigen-3-X/antigen-2-Y but not the combination ofantigen-3-X/antigen-2-X was able to inhibit anti-IgM induced CD86expression on B cells after 24 hours (see FIG. 14). The IC₅₀, asextrapolated using a 4 parameter logistic curve fit using Graphpad Prism6, was 10.3 nM (the data represents mean values and the error bars arestandard deviations).

Example 10 the Inhibitory Effect of an Anti-Antigen 4 and anAnti-Antigen 2 can Only Reproduced when the Antibodies are Arranged in aBispecific Orientation

Human PBMC were prepared according to general method 1. During thisperiod combinations of bispecific, bivalent or mixtures of antibodieswere created by diluting equimolar (200 nM) quantities of Fab′A-X(Fab-scFv) and/or Fab′B-Y (Fab-peptide) with antigen specificity for thecell surface proteins antigen 4 and antigen 2 in DMEM containing 10%calf serum and 2 mM glutamine. These combinations are shown in Table 10.

TABLE 10 Grid of bispecific, bivalent or mixtures with specificity forantigen 4 & 2 (B-Y) Fab B (A-X) Fab A antigen 2-X antigen 2-Y antigen4-Y 2-X:Y-4 4-Y Y-2 antigen 2-X — 2-X:Y-2

where X is a scFv (52SR4) and Y is a peptide (GCN4)

Fab′A-X and/or Fab′B-Y were incubated according to general method 2.Cells were then stained according to general method 3.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of Akt and PLCg2 levels were calculated for each well.All data was then expressed as the percentage inhibition of the maximalresponse (anti-IgM only) minus the background (cells only).

FIGS. 15 and 16 show that only the bispecific combination of ananti-antigen 4 (VR4248) and an anti-antigen 2 (VR4246) but not themixtures of anti-antigen 4 (VR4248) and anti-antigen 2 (VR4246)antibodies inhibited phosphorylated Akt and PLCg2 expression (the datarepresents mean values and the error bars are 95% confidence intervals).

In order to validate the inhibition seen with the bispecific combinationof an anti-antigen 4 (VR4248) and an anti-antigen 2 (VR4246), thiscombination was titrated in an assay system measuring anti-IgM inducedCD86 expression on B cells.

As can be seen in FIG. 17, a titration of the combination of antigen4-X/antigen 2-Y was able to inhibit anti-IgM induced CD86 expression onB cells after 24 hours. The IC₅₀, as extrapolated using a 4 parameterlogistic curve fit using Graphpad Prism 6, was 4.7 nM (the datarepresents mean values and the error bars are standard deviations).

Example 11 Bispecific Complex Characterisation

Purification of Functional Screening Reagents:

The functional screening formats Fab-X (Fab-scFv-His) and Fab-Y(Fab-peptide-His) were purified as follows after standard CHOexpression. Clarified cell culture supernatants were 0.22 μm sterilefiltered using a 1 L stericup. The pH was measured and where necessaryadjusted to pH7.4. The prepared supernatants were loaded at 5 ml/minonto 5 ml HisTrap Nickel Excel (GE Healthcare) columns equilibrated in10 mM Sodium phosphate, 0.5 M NaCl, pH7.4. The columns were washed with15 mM imidazole, 10 mM Sodium phosphate, 0.5M NaCl, pH7.4 and theneluted with 250 mM imidazole, 10 mM Sodium phosphate, 0.5M NaCl, pH7.4.The elution was followed by absorbance at 280 nm and the elution peakcollected. The peak elutions were analysed by size exclusionchromatography on a TSKgel G3000SWXL; 5 μm, 7.8×300 mm column developedwith an isocratic gradient of 0.2M phosphate, pH7.0 at 1 ml/min, withdetection by absorbance at 280 nm. Samples of sufficient purity wereconcentrated to >1 m/ml and diafiltered into PBS pH7.4 (Sigma AldrichChemicals) using Amicon Ultra-15 concentrators with a 10 kDa molecularweight cut off membrane and centrifugation at 4000×g in a swing outrotor. Where product quality was not sufficient the nickel columnelutions were concentrated and applied to either a XK16/60 or XK16/60Superdex200 (GE Healthcare) column equilibrated in PBS, pH7.4 (SigmaAldrich Chemicals). The columns were developed with an isocraticgradient of PBS, pH7.4 (Sigma Aldrich Chemicals) at 1 ml/min or 2.6ml/min respectively. Fractions were collected and analysed by sizeexclusion chromatography on a TSKgel G3000SWXL; 5 μm, 7.8×300 mm columndeveloped with an isocratic gradient of 0.2 M phosphate, pH7.0 at 1ml/min, with detection by absorbance at 280 nm. Selected fractions werepooled and concentrated to >1 mg/ml using an Amicon Ultra-15concentrator with a 10 kDa molecular weight cut off membrane andcentrifugation at 4000×g in a swing out rotor.

Analysis of Bispecific Formation in Solution

Experiment 1

Purified Fab-X (VR4247) and purified Fab-Y (VR4248) were mixed in a oneto one molar ratio, with a total protein concentration of 500 μg/ml andincubated overnight at ambient temperature. Controls consisted of theindividual parts of the mixture at the same concentration as they wouldbe in the mixture. 100 μl of the sample and each control was injectedonto a TSKgel G3000SWXL; 5 μm, 7.8×300 mm column developed with anisocratic gradient of 0.2 M phosphate, pH7.0 at 1 ml/min. Detection wasby absorbance at 280 nm (see FIG. 18).

The size exclusion chromatograms in FIG. 18 show that the Fab-X (VR4247)control has a main peak of 92% of the total peak area with a retentiontime of 8.610 metric minutes. The Fab-Y (VR4248) control has a main peakof 94% of the total peak area with a retention time of 10.767 metricminutes. The retention times measured for the Fab-X and Fab-Y controlswere converted to apparent molecular weight of 95 kDa and 35 kDarespectively by using a standard curve created from the retention timesof BioRad gel filtration standards (151-1901) run under the sameconditions. These apparent molecular weights are consistent with theexpected apparent molecular weights for Fab-scFv and Fab-peptidemolecules. The main peak for the Fab-X (VR4247)/Fab-Y (VR4248) mixturehas a retention time of 9.289 metric minutes. This is converted as aboveto an apparent molecular weight of 187 kDa. This apparent molecularweight is consistent with that expected for the pairing of one Fab-X(VR4247) with one Fab-Y (VR4248). The main peak is also 84% of the totalpeak area suggesting that most of the Fab-X (VR4247) and Fab-Y (VR4248)have formed the 1 to 1 bispecific protein complex. The small additionalshoulder and peak that elute after the main peak are consistent with theFab-X (VR4247) and Fab-Y (VR4248) starting materials.

Experiment 2

Purified Fab-X (VR4130) and Fab-Y (VR4131) were mixed in a one to onemolar ratio, with a total protein concentration of 500 μg/ml. Aliquotsof this mixture were then diluted with PBS pH7.4 to a concentration of50 μg/ml and 5 μg/ml. Controls consisting of the individual parts of themixture at the same concentration as they would be in the 500 μg/mlmixture were also set up. All mixtures and controls were incubatedovernight at ambient temperature. 100 μl of all samples and controlswere injected onto a TSKgel G3000SWXL; 5 μm, 7.8×300 mm column developedwith an isocratic gradient of 0.2M phosphate, pH7.0 at 1 ml/min.Detection was by absorbance at 214 nm (see FIG. 19, FIG. 20 and Table11).

The size exclusion chromatograms in FIG. 19 show that the Fab-X (VR4130)control has a main peak of 91% of total peak area with a retention timeof 8.634 metric minutes. The Fab-Y (VR4131) control has a main peak of97% of total peak area with a retention time of 9.361 metric minutes.The retention times measured for the Fab-X and Fab-Y controls wereconverted to apparent molecular weights of 109 kDa and 55 kDarespectively by using a standard curve created from the retention timesof BioRad gel filtration standards (151-1901) run under the sameconditions. These apparent molecular weights are consistent with theexpected apparent molecular weights for Fab-scFv and Fab-peptidemolecules. The main peak for the Fab-X (VR4130)/Fab-Y (VR4131) mixturehas a retention time of 8.016 metric minutes. This was converted asabove to an apparent molecular weight of 198 kDa. This apparentmolecular weight is consistent with that expected for the pairing of oneFab-X (VR4130) with one Fab-Y (VR4131). The main peak is also 82% of thetotal peak area suggesting that most of the Fab-X (VR4130) and Fab-Y(VR4131) have formed the 1 to 1 complex. The two small peaks that eluteafter the main peak are consistent with the Fab-X (VR4130) and Fab-Y(VR4131) starting materials.

The size exclusion chromatograms in FIG. 20 are for the Fab-X(VR4130)/Fab-Y (VR4131) 1 to 1 mixtures at 500 μg/ml, 50 μg/ml and 5μg/ml concentration. All the traces are similar with corresponding peaksbetween samples having similar retention times and similar relative peakheights and areas. The percentage peak area is collated in Table 11,where the % of each peak remains fairly constant upon dilution of themixture. This indicates that the Fab-X/Fab-Y 1 to 1 complex remains as acomplex at all the concentrations tested. 75% of the Fab-X and Fab-Y arepresent as the 1 to 1 complex even when the mixture is diluted to 5μg/ml which is equivalent to concentration of 40 nM for the complex.

TABLE 11 Size exclusion peak area data for Fab-X (VR4130)/Fab-Y (VR4131)1:1 molar ratio mixtures at 500 μg/ml, 50 μg/ml and 5 μg/ml. Peaks weredetected at an absorbance of 214 nm. % Peak Area Fab-X (VR4130)/Concentrations Fab-Y (VR4131) Fab-X Fab-Y μg/ml nM 1 to 1 complex(VR4130) (VR4131) 500 4000 82%  4% 5% 50 400 81% 11% 3% 5 40 75% 21% 3%

Hence, the results of these experiments indicate that a high proportionof the Fab-X and Fab-Y fusion proteins form the desired bispecificcomplexes, with a minimal proportion of monomers left over and noevidence of homodimer formation.

Example 12: Grid Screening of Large Panels of Heterodimerically TetheredProtein Complexes to Identify Novel Bispecific Antibody Targets

Introduction: Following the successful validation of the bispecificformat and screening method in the earlier examples the screening wasexpanded to a larger number of antigen pairs. A panel of antibodyvariable (V) region pairs to 23 different antigens expressed on B cellswas generated. Using the Fab-Kd-Fab [i.e. A-X:Y-B wherein A and B areFab fragments] format a grid of heterodimerically tethered proteincomplexes was formed representing multiple V region combinations of eachof 315 different antigen pair combinations. These combinations werescreened for their ability to modulate BCR (B cell receptor) signallingin a high through-put flow cytometry assay to select novel target pairsfor intervention with a bispecific antibody.

Immunisation:

DNA encoding selected antigens was obtained by gene synthesis orcommercial sources & cloned into an expression vector with a strongconstitutive promoter. Plasmid DNA was then transfected into Rab-9rabbit fibroblast cells (ATCC® CRL-1414™) using an in-houseelectroporation system. Twenty four hours later cells were checked forantigen expression by flow cytometry & frozen in aliquots in liquidnitrogen until use. Up to 6 antigens were immunised per rabbit by eitherco-expression on the same cell or making mixtures of singly or multipletransfected cells. Rabbits were immunised with 3 doses of cells.

Antibody Discovery:

B cell cultures were prepared using a method similar to that describedby Zubler et al. (1985). Briefly, spleen or PBMC-derived B cells fromimmunized rabbits were cultured at a density of approximately 2000-5000cells per well in bar-coded 96-well tissue culture plates with 200μl/well RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (PAAlaboratories ltd), 2% HEPES (Sigma Aldrich), 1% L-Glutamine (Gibco BRL),1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol(Gibco BRL), 3% activated splenocyte culture supernatant andgamma-irradiated mutant EL4 murine thymoma cells (5×10⁴/well) for sevendays at 37° C. in an atmosphere of 5% CO₂.

The presence of antigen-specific antibodies in B cell culturesupernatants was determined using a homogeneous fluorescence-basedbinding assay using HEK293 cells co-transfected with the antigens thatthe rabbits were immunized with. Screening involved the transfer of 10ul of supernatant from barcoded 96-well tissue culture plates intobarcoded 384-well black-walled assay plates containing HEK293 cellstransfected with target antigen (approximately 3000 cells/well) using aMatrix Platemate liquid handler. Binding was revealed with a goatanti-rabbit IgG Fcγ-specific Cy-5 conjugate (Jackson). Plates were readon an Applied Biosystems 8200 cellular detection system.

Following primary screening, positive supernatants were consolidated on96-well bar-coded master plates using an Aviso Onyx hit-picking robotand B cells in cell culture plates frozen at −80° C. Master plates werethen screened in a homogeneous fluorescence-based binding assay onHEK293 cells transfected with antigens separately and SUPERAVIDIN™ beads(Bangs Laboratories) coated with recombinant protein as a source ofantigen. This was done in order to determine the antigen specificity foreach well.

To allow recovery of antibody variable region genes from a selection ofwells of interest, a deconvolution step was performed to enableidentification of the antigen-specific B cells in a given well thatcontained a heterogeneous population of B cells. This was achieved usingthe Fluorescent foci method (Clargo et al., 2014.Mabs 2014 Jan. 1: 6(1)143-159; EP1570267B1). Briefly, Immunoglobulin-secreting B cells from apositive well were mixed with either HEK293 cells transfected withtarget antigen or streptavidin beads (New England Biolabs) coated withbiotinylated target antigen and a 1:1200 final dilution of a goatanti-rabbit Fcγ fragment-specific FITC conjugate (Jackson). After staticincubation at 37° C. for 1 hour, antigen-specific B cells could beidentified due to the presence of a fluorescent halo surrounding that Bcell. A number of these individual B cell clones, identified using anOlympus microscope, were then picked with an Eppendorf micromanipulatorand deposited into a PCR tube. The fluorescent foci method was also usedto identify antigen-specific B cells from a heterogeneous population ofB cells directly from the bone marrow of immunized rabbits.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested secondary PCR incorporating restriction sites at the 3′ and 5′ends allowing cloning of the variable region into mouse Fab-X and Fab-Y(VH) or mouse kappa (VL) mammalian expression vectors. Heavy and lightchain constructs for the Fab-X and Fab-Y expression vectors wereco-transfected into HEK-293 cells using Fectin 293 (Life Technologies)or Expi293 cells using Expifectamine (Life Technologies) and recombinantantibody expressed in 6-well tissue culture plates in a volume of 5 ml.After 5-7 days expression, supernatants were harvested. Supernatantswere tested in a homogeneous fluorescence-based binding assay on HEK293cells transfected with antigen and SUPERAVIDIN™ beads (BangsLaboratories) coated with recombinant protein or antigen transfected HEKcells. This was done to confirm the specificity of the clonedantibodies.

Production of Small Scale Fab A-X and Fab B-Y (Small Scale (50 mL)Expi293 Transfection)

The Expi293 cells were routinely sub-cultured in EXPI293™ ExpressionMedium to a final concentration of 0.5×10⁶ viable cells/mL and wereincubated in an orbital shaking incubator (Multitron, Infors HT) at 120rpm 8% CO₂ and 37° C.

On the day of transfection cell viability and concentration weremeasured using an automated Cell Counter (Vi-CELL, Beckman Coulter). Toachieve a final cell concentration of 2.5×10⁶ viable cells/mL theappropriate volume of cell suspension was added to a sterile 250 mLErlenmeyer shake flask and brought up to the volume of 42.5 mL by addingfresh, pre-warmed EXPI293™ Expression Medium for each 50 mLtransfection.

To prepare the lipid-DNA complexes for each transfection a total of 50μg of heavy chain and light chain plasmid DNAs were diluted in OPTI-MEM®I medium (LifeTechnologies) to a total volume of 2.5 mL and 135 μL ofEXPIFECTAMINE™ 293 Reagent (LifeTechnologies) was diluted in OPTI-MEM® Imedium to a total volume of 2.5 mL. All dilutions were mixed gently andincubate for no longer than 5 minutes at room temperature before eachDNA solution was added to the respective diluted EXPIFECTAMINE™ 293Reagent to obtain a total volume of 5 mL. The DNA-EXPIFECTAMINE™ 293Reagent mixtures were mixed gently and incubated for 20-30 minutes atroom temperature to allow the DNA-EXPIFECTAMINE™ 293 Reagent complexesto form.

After the DNA-EXPIFECTAMINE™ 293 reagent complex incubation wascompleted, the 5 mL of DNA-EXPIFECTAMINE™ 293 Reagent complex was addedto each shake flask. The shake flasks were incubated in an orbitalshaking incubator (Multitron, Infors HT) at 120 rpm, 8% CO₂ and 37° C.

Approximately 16-18 hours post-transfection, 250 μL of EXPIFECTAMINE™293 Transfection Enhancer 1 (LifeTechnologies) and 2.5 mL ofEXPIFECTAMINE™ 293 Transfection Enhancer 2 (LifeTechnologies) were addedto each shake flask.

The cell cultures were harvested 7 days post transfection. The cellswere transferred into 50 mL spin tubes (Falcon) and spun down for 30 minat 4000 rpm followed by sterile filtration through a 0.22 um Stericup(Merck Millipore). The clarified and sterile filtered supernatants werestored at 4° C. Final expression levels were determined by ProteinG-HPLC.

Small Scale (50 ml) Purification:

Both Fab-X and Fab-Y were purified separately by affinity capture usinga small scale vacuum based purification system. Briefly, the 50 ml ofculture supernatants were 0.22 μm sterile filtered before 500 μL of NiSepharose beads (GE Healthcare) were added. The supernatant beadsmixture was then tumbled for about an hour before supernatant wasremoved by applying vacuum. Beads were then washed with Wash 1 (50 mMSodium Phosphate 1 M NaCl pH 6.2) and Wash 2 (0.5 M NaCl). Elution wasperformed with 50 mM sodium acetate, pH4.0+1M NaCl. The eluted fractionsbuffer exchanged into PBS (Sigma), pH7.4 and 0.22 μm filtered. Finalpools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE(reduced & non-reduced) and for endotoxin using the PTS Endosafe system.

Screening Assays

Donor PBMCs were rapidly thawed using a water bath set to 37° C., andcarefully transferred to a 50 ml Falcon tube. They were then diluteddropwise to 5 ml in assay media to minimise the osmotic shock. The cellswere then diluted to 20 ml carefully before adding the final mediadiluent to make the volume 50 ml. The cells were then spun at 500 g for5 minutes before removing the supernatant and resuspending the cells in1 ml media. The cells were then counted and diluted to 1.66×10⁶ cells/mlbefore dispensing 30 μl per well into a V-bottom TC plate giving a finalassay concentration of 5.0×10⁴ cells/well. The cell plate was thenstored covered in a 37° C., 5% CO₂ incubator until they were required,giving them a minimum of 1 hour to rest.

Fab-X and Fab-Y reagents were mixed in an equimolar ratio at 5× thefinal assay concentration in assay media and incubated for 90 min at 37°C., 5% CO₂. Samples were prepared in a 96-well U-bottom polypropyleneplate and covered during the incubation. 10 μl of 5×Fab-KD-Fab mixturewas added to the appropriate test wells containing cells and mixed byshaking at 1000 rpm for 30 sec prior to being incubated for 90 min at37° C., 5% CO₂.

The cells were then stimulated with 10 μl of anti-human IgM. The finalassay concentration of stimulus varied depending on the assay panelreadouts, the three antibody cocktails A, B and C (detailed below) werestimulated at a final assay concentration of either 50 μg/ml (cocktail A& C) or 25 μg/ml (cocktail B). The assay plates were then gently mixedat 1000 rpm for 30 sec prior to incubation at 37° C., 5% CO₂ for 5 min(antibody cocktail A & C) or 2 min (antibody cocktail B). The assay wasstopped by adding 150 μl ice-cold BD CytoFix to all wells and incubatedfor 15 min at RT. The fixed cells were then spun at 500 g for 5 min topellet the cells and allow removal of the supernatant using a BioTekELx405 plate washer. The pellet was re-suspended by vortexing the plateat 2400 rpm for 30 sec. The cells were then permeabilised at 4° C. byadding 100 μl ice-cold BD Cell Permeabilisation Buffer III for 30 min.The cells were then washed in 100 μl FACS buffer and spun at 500 g for 5min. Supernatant was again removed by the ELx405 before using it torapidly dispense 200 μl FACS Buffer to wash away any residualpermeabilisation buffer. Cells were again spun at 500 g and thesupernatant removed by inversion. During the preceding spin step theantibody cocktail was prepared in FACS Buffer and kept shielded from thelight. The cells were then re-suspended by vortexing (2400 RPM, 30 sec)before 20 μl of antibody cocktail was added to all wells and the plateshaken for 30 sec at 1000 rpm. The cells were then incubated for 60 minat RT in the dark.

The cells were then washed twice in 200 μl FACS buffer with a 500 g spinand supernatant removed after each step. Finally the cells werere-suspended by vortexing for 30 sec at 2400 rpm before adding a final20 μl FACS buffer. The plate(s) were then read on the IntellicytHTFC/iQue instrument.

FACS Buffer=PBS+1% BSA+0.05% NaN₃+2 mM EDTA

Antibody Cocktail A=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5 PLCγ2AF88+1:10 Akt AF647+1:50 ERK1/2 PE (diluted in FACS buffer).

Antibody Cocktail B=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5 Syk PE+1:5BLNK AF647 (diluted in FACS buffer)

Antibody Cocktail C=1:5 CD20 PerCp-Cy5.5 (Biolegend)+1:5 PLCγ2AF488+1:10 Akt AF647+1:5 Syk PE (diluted in FACS buffer)

Reagent Supplier Catalogue number Anti-human IgM Southern Biotech2022-14 CytoFix BD Biosciences 554655 Perm Buffer III BD Biosciences558050 Anti Akt (pS473) AF647 BD Biosciences 561670 Anti SYK (pY348) PEBD Biosciences 558529 Anti PLCγ2 (pY759) AF488 BD Biosciences 558507Anti-BLNK(pY84) AF647 BD Biosciences 558443 Anti ERK1/2 (pT202/pY204) PEBD Biosciences 561991 Anti-human CD20 PerCp-Cy5.5 BD Biosciences 558021Anti-human CD20 AF488 BD Biosciences 558056 Anti-human CD20 PerCp-Cy5.5Biolegend 340508 Phosphate Buffer Saline (PBS) Fisher Scientific10562765 RPMI 1640 Life Technologies 31870 Foetal Calf Serum (FCS) LifeTechnologies 16140 Glutamax Life Technologies 35050Penicillin/Streptomycin (P/S) Life Technologies 15070 EDTA Sigma 03690Sodium Azide (NaN3) Sigma S2002 Bovine Serum Albumin (BSA) Sigma A1470

Fab-X+Fab-Y combinations were screened with either antibody cocktail Aand B or C alone. All screens were conducted on cone cells from 2different blood donors. Data was captured and evaluated usingcommercially available software tools. A total of 2500 Fab-X+Fab-Ycombinations were screened to 315 different antigen combinations.

Results

The percentage inhibition of the induction of phosphorylation of BCRsignalling cascade proteins by each Fab-Kd-Fab [i.e. A-X:Y-B where A andB are Fab fragments] combination was calculated, in this example lookingfor new combinations of antigens that inhibit B cell function, thecriteria for a positive combination was set as at least 30% inhibitionof at least two phospho-readouts by at least one combination of Vregions. According to this threshold 11 new antigen pair combinationsout of 315 examined met the required criteria. This represents a 3.5%hit rate demonstrating the importance of screening large numbers ofcombinations to find those of desired activity.

FIGS. 21-23 show the data for the antigen grid cross specificities.Values are percentage inhibition (negative value for activation) ofphosphorlylation of Syk, PLCg2 & AKT respectively and represent the meanof multiple V-region combinations evaluated. 315 different antigencombinations were tested and as can be seen the effect on BCR signallingby different combinations of antibody varied significantly from stronginhibition e.g. antigen 2 on Fab-X combined with antigen 3 and 4 onFab-Y (69.66% and 70.4% inhibition of phospho Syk FIG. 21) to activatione.g antigen 6 on X and antigen 11 on Y (minus 118.10% phospho Syk FIG.21).

Each data point representing the mean % values represented in FIGS.21-23 is shown for antigen 2 on Fab-X and antigen 3 on Fab-Y in FIG. 24.In this case, 23 different combinations of different antibody V regionswere evaluated. The same antigen combination but in alternativeorientation, i.e. antigen 2 on Fab-Y and antigen 3) on Fab-X is shown inFIG. 25. In this case, 9 different combinations of different antibodyV-regions were evaluated. All V regions show inhibition butadvantageously this method can also be used in the selection of optimalV-region combinations.

Similarly, each data point representing the mean % values represented inFIGS. 21-23 is shown for antigen combination 2 on Fab-X and antigen 4 onFab-Y in FIG. 26. In this case, 10 different combinations of differentantibody V regions were evaluated. The same antigen combination but inalternative orientation, i.e. antigen 2 on Fab-Y and antigen 4 on Fab-Xis shown in FIG. 27. In this case, 6 different combinations of differentantibody V regions were evaluated. Again, all V regions show inhibitionbut optimal V region combinations can be identified and selected usingthe method.

Example 13—Evaluation of Transiently Expressed HeterodimericallyTethered Protein Complexes to Evaluate Whether FabA-X:Y-FabB GridScreening can Identify Novel Bispecific Antibody Targets withoutRecourse to Protein Purification

Introduction:

V-regions to 2 different antigens, 2 and 3 that inhibit B cellsignalling as a bispecific antibody which were identified using theFab-Kd-Fab [FabA-X:Y-FabB] format and grid screening ofheterodimerically tethered protein complexes were expressed transientlyas FabA-X and FabB-Y. The activity of transiently expressed (withoutsubsequent purification) and a purified FabA-X and FabB-Y combinationwas compared to evaluate whether grid screening could be conducted withthe direct products of transient expression instead of purifiedcomponents.

Immunisation:

The preparation of antigen expressing cells and immunisation of rabbitswas carried out in the same way as described in Example 12.

Antibody Discovery B cell cultures were prepared in the same way asdescribed in Example 12.

The screening of antigen-specific antibodies in B cell culturesupernatants and the deconvolution step for identification of antigenspecific B cells was determined in the same way as described in Example12.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested 2° PCR incorporating restriction sites at the 3′ and 5′ endsallowing cloning of the variable region into mouse Fab-X and Fab-Y (VH)or mouse kappa (VL) mammalian expression vector. A 3° PCR was thenperformed enabling the combination of amplified variable regions, humanCMV promoter fragment and rabbit gamma 1 heavy constant or rabbit kappaconstant fragment to generate separate heavy and light transcriptionallyactive PCR (TAP) fragments. These DNA fragments were used directly forrecombinant expression of rabbit full-length IgG antibodies in HEK-293cells using 293Fectin (Life Technologies) or in Expi293 cells usingExpifectamine (Life Technologies). The resulting recombinant antibodieswere then screened for antigen binding using a homogeneousfluorescence-based binding assay on HEK-293 cells transfected withantigen and SUPERAVIDIN™ beads (Bangs Laboratories) coated withrecombinant protein. Once specificity was confirmed with the TAPtransients, antibody genes were cloned into Fab-X and Fab-Y expressionvectors. Heavy and light chain constructs were co-transfected intoHEK-293 cells using Fectin 293 (Life Technologies) or Expi293 cellsusing Expifectamine (Life Technologies) and recombinant antibodyexpressed in 6-well tissue culture plates in a volume of 5 ml. After 5-7days expression, supernatants were harvested. Supernatants were testedin a homogeneous fluorescence-based binding assay on HEK293 cellstransfected with antigen and SUPERAVIDIN™ beads (Bangs Laboratories)coated with recombinant protein or antigen transfected HEK cells. Thiswas done to confirm the specificity of the cloned antibodies.

Production of Transient Supernatants Containing Fab-X and Fab-Y

The same Expi293 transfection method described in Example 12 was used toproduce the transient supernatants containing Fab-X and Fab-Y.

Production of Purified Fab-X and Fab-Y

Suspension CHOSXE cells were pre-adapted to CDCHO media (Invitrogen)supplemented with 2 mM (100×) glutamx. Cells were maintained inlogarithmic growth phase agitated at 140 rpm on a shaker incubator(Kuner AG, Birsfelden, Switzerland) and cultured at 37° C. supplementedwith 8% CO₂.

Prior to transfection, the cell numbers and viability were determinedusing CEDEX cell counter (Innovatis AG. Bielefeld, Germany) and requiredamount of cells (2×10⁸ cells/ml) were transferred into centrifugeconical tubes and were spun at 1400 rpm for 10 minutes. The Pelletedcells were re-suspended in sterile Earls Balanced Salts Solution andspun at 1400 rpm for further 10 minutes. Supernatant was discarded andpellets were re-suspended to desired cell density.

Vector DNA at a final concentration of 400 ug for 2×10⁸ cells/ml mix and800 μl was pipetted into Cuvettes (Biorad) and electroporated usingin-house electroporation system.

Transfected cells were transferred directly into 1λ3 L Erlenmeyer Flaskscontained ProCHO 5 media enriched with 2 mM glutamx and antibioticantimitotic (100×) solution (1 in 500) and Cells were cultured in Kuhnershaker incubator set at 37° C., 5% CO₂ and 140 rpm shaking. Feedsupplement 2 g/L ASF (AJINOMOTO) was added at 24 hr post transfectionand temperature dropped to 37° C. for further 13 days culture. At dayfour 3 mM sodium buryrate (n-butric acid sodium salt, Sigma B-5887) wasadded to the culture.

On day 14, cultures were transferred to tubes and supernatant separatedfrom the cells after centrifugation for 30 minutes at 4000 rpm. Retainedsupernatants were further filtered through 0.22 μm SARTOBRAN® PMillipore followed by 0.22 μm Gamma gold filters. Final expressionlevels were determined by Protein G-HPLC.

The Fab-X and Fab-Y were purified by affinity capture using the AKTAXpress systems and HisTrap Excel pre-packed nickel columns (GEHealthcare). The culture supernatants were 0.22 μm sterile filtered andpH adjusted to neutral, if necessary, with weak acid or base beforeloading onto the columns. A secondary wash step, containing 15-25 mMImidazole, was used to displace any weakly bound host cellproteins/non-specific His binders from the nickel resin. Elution wasperformed with 10 mM sodium phosphate, pH7.4+1 M NaCl+250 mM imidazoleand 2 ml fractions collected. One column volume into the elution thesystem was paused for 10 minutes to tighten the elution peak, andconsequently decrease the total elution volume. The cleanest fractionswere pooled and buffer exchanged into PBS (Sigma), pH7.4 and 0.22 μmfiltered. Final pools were assayed by A280 Scan, SE-HPLC (G3000 method),SDS-PAGE (reduced & non-reduced) and for endotoxin using the PTSEndosafe system.

Functional Assays

Activation Marker Assay:

Antigen 2-specific Fab′-Y and antigen 3-specific Fab′-X, either purifiedor in transient supernatant, were incubated together for 60 minutes (ina 37° C. & 5% CO₂ environment) at equimolar concentration. Thecombinations were titrated from a starting molarity of 185 nM, in 1:4serial dilutions. A mock supernatant was also included, titrated fromneat. In V-bottomed 96 well plates, 1.5×10⁵ PBMC were added to wells, towhich were added titrated Fab′-X and Fab′-Y combinations or mocksupernatant. The combinations and cells were then incubated together fora further 90 minutes. After this time B cells were activated by theaddition of 12.5 μg/mL of goat F(ab′)2 anti-human IgM (SouthernBiotechnology) for 24 hours at 37° C. plus 5% CO₂.

To the wells were added 100 μL, ice-cold FACS buffer (PBS+1% BSA+0.1%NaN₃+2 mM EDTA), the plates were sealed and covered with wet-ice forapproximately 15 minutes, before centrifuging at 500×g for 5 minutes at4° C. Excess supernatant was discarded from the cell pellets and theplates shaken at 2000 rpm for 30 seconds.

Cells were then stained with a cocktail of fluorescently labelledanti-CD19, anti-CD20 and anti-CD71 antibodies (BD Biosciences). Plateswere shaken briefly and incubated for 1 hour on wet-ice in the dark.After this time plates were washed twice and resuspended in 20 μL ofFACS buffer. Cellular expression of CD19, CD20 and CD71 was measuredusing an Intellicyt IQUE® Screener flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of CD71 levels were calculated for each well. All datawas then expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only).

PhosFlow Assay:

Antigen 2-specific Fab′-Y and antigen 3-specific Fab′-X, either purifiedor in transient supernatant, were incubated together for 60 minutes (ina 37° C. & 5% CO₂ environment) at equimolar concentration. Thecombinations were titrated from a starting molarity of 185 nM, in 1:4serial dilutions. A mock supernatant was also included, titrated fromneat. In V-bottomed 96 well plates, 5.0×10⁴ PBMC were added to wells, towhich were added titrated Fab′-X and Fab′-Y combinations or mocksupernatant. The combinations and cells were then incubated together fora further 90 minutes. After this time B cells were activated by theaddition of 25 μg/mL of goat F(ab′)2 anti-human IgM (SouthernBiotechnology) for 15 minutes at 37° C. plus 5% CO₂. The signallingreaction was then halted by adding an equal volume of Cytofix buffer (BDBiosciences). Plates were then left at room temperature for 15 minutesbefore centrifugation at 500×g for 5 minutes. Excess supernatant wasdiscarded from the cell pellet which was resuspended in FACS buffer(PBS+1% BSA+0.01% NaN₃+2 mM EDTA) and washed once more. Cells were thenresuspended in ice cold Perm Buffer III (BD Biosciences) for 30 minutesbefore being washed twice in flow buffer. Cells were then stained with afluorescently labelled anti-CD20 antibody (BD Biosciences) and ananti-phosphorylated p38 antibody that recognises the conserved dualphosphorylated site pT180/pY182. Plates were then resuspended andincubated for 1 hour at room temperature in the dark. After this timeplates were washed a further two times and resuspended in 20 μL of FACSbuffer. Cellular expression of CD20 and phosho-p38 was measured using anIntellicyt IQUE® flow cytometer.

Using the data analysis software package FORECYT™ (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of p38 levels were calculated for each well. All data wasthen expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only).

Results

Activation Marker Assay:

As can be seen in FIG. 28, the data shows that the combination ofAntigen 3 with Antigen 2 whether purified or from transient supernatant,can inhibit CD71 expression on B-cells stimulated with anti-IgM.

PhosFlow Assay:

The data in FIG. 29 shows that the combination of Antigen 3 with Antigen2 whether purified or from transient supernatant, can inhibitphosphorylated p38 in B-cells stimulated with anti-IgM.

The surprising ability to be able to construct the bispecific complexesof the present invention directly from transiently expressed cultureswithout recourse to purification allows even higher throughput screeningof bispecific complexes to be achieved than when purified components areused.

Example 14—Screening of Transiently Expressed V-Regions to Antigen 3 asFab-X with Purified Anti-Antigen 2 Fab-Y in Heterodimerically TetheredProtein Complexes to Select Optimal Antigen 3 Antibody V-Regions

Introduction:

New V-regions to Antigen 3 that inhibit B cell signalling as abispecific antibody in combination with Antigen 2 specific V regionswere identified using grid screening of heterodimerically tetheredprotein complexes. The Antigen 3 V regions were expressed transiently asFab-X and combined with purified ant-antigen 2 Fab-Y. The inhibition ofactivation of B cell signalling was measured to select the most potentAntigen 3 and antigen 2 V regions.

The preparation of antigen expressing cells and immunisation of rabbitswas carried out in the same way as described in Example 12.

Antibody Discovery:

B cell cultures were prepared in the same way as described in Example12.

The screening of antigen-specific antibodies in B cell culturesupernatants and the deconvolution step for identification of antigenspecific B cells was determined in the same way as Example 12.

Additional variable regions were discovered by the direct foci methoddirectly from spleen and bone marrow-derived B cells of immunized mice.Briefly, cells at a final density of between 4×10⁵ and 8×10⁵ cells/mlwere mixed with streptavidin beads (New England Biolabs) coated withbiotinylated antigenand a 1:1200 final dilution of goat anti-mouse Fcγfragment-specific FITC conjugate (Jackson). After static incubation at37° C. for 1 hour, antigen-specific B cells could be identified due tothe presence of a fluorescent halo surrounding that B cell. A number ofthese individual B cell clones, identified using an Olympus microscope,were then picked with an Eppendorf micromanipulator and deposited into aPCR tube.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested 2° PCR incorporating restriction sites at the 3′ and 5′ endsallowing cloning of the variable region into mouse Fab-X and mouse kappa(VL) mammalian expression vector. These vectors were then co-transfectedin HEK-293 cells using 293Fectin (Life Technologies) or in Expi293 cellsusing Expifectamine (Life Technologies) and left to express for 6 days.Supernatants were tested in a homogeneous fluorescence-based bindingassay on HEK293 cells transfected with antigen and SUPERAVIDIN™ beads(Bangs Laboratories) coated with recombinant protein or antigentransfected HEK cells. This was done to confirm the specificity of thecloned antibodies.

In addition to the Fab-X transient supernatants, negative control Mocksupernatants were prepared in the same way using an irrelevant controlDNA.

The expression levels of Fab-X were determined by Protein G-HPLC.

Production of Purified Fab-Y:

Purified Fab-Y was prepared using the same method described in Example13

Functional Assay

The same functional assay as described in Example 12 was used, exceptthat instead of 3 different antibody cocktails, only one cocktail wasused with the same assay concentrations and incubation conditions asdescribed for antibody cocktail A in Example 12.

Antibody Cocktail=1:3 CD20 PerCp-Cy5.5+1:5 PLCγ2 AF88+1:10 Akt AF647+1:5p38 MAPK PE (diluted in FACS buffer).

Results

As can be seen in FIGS. 30-33, the data shows that the combination ofdifferent transiently expressed antigen 3 mouse V regions in Fab-X with2 different purified antigen 2 V regions (VR447 and VR4450) in Fab-Y caninhibit B cell activation to different levels and screening thereforefacilitates selection of optimal V regions. Combinations with transientFab-X are compared to a reference combination with a purified Fab-X(VR4126).

Example 15—Comparison of the Activity of Antigen 2 Plus Antigen 3Co-Targeting in Fab-Kd-Fab Screening Format to a Molecularly LinkedBispecific BYbe Format

Introduction:

To check that target pair activity identified in the Fab-Kd-Fabheterodimerically tethered screening complex could translate to similardesired activity in an alternative therapeutic molecularly linkedformat, Antigen 2 specificity (VR4447) and antigen 3 specificity(VR4130) were generated in a BYbe format. This BYbe format consists ofthe anti-Antigen 3 V regions (VR4130) as a disulphide stabilised (ds)single chain (sc)-Fv fused to the heavy chain of the anti-Antigen 2 Fab(VR4447).

Methods:

As described in Example 13 except the purification of BYbes forfunctional screening was performed as follows:

The functional screening BYbe (Fab-dsscFv [scFv off C-terminus of Fabheavy chain]) formats were purified as follows. Clarified cell culturesupernatants from standard expiHEK or CHO expression were 0.22 μmsterile filtered. The filtered supernatants were loaded at 2 ml/min onto50 ml GammabindPlus Sepharose XK26 columns (GE Healthcare) equilibratedin PBS pH7.4 (Sigma Aldrich Chemicals). After loading the columns werewashed with PBS pH7.4 and then eluted with 0.1M Glycine/HCl. pH2.7. Theelution was followed by absorbance at 280 nm, the elution peakcollected, and then neutralised with 1/25^(th) volume of 2M Tris/HClpH8.5. The neutralised samples were concentrated using Amicon Ultra-15concentrators with a 10 kDa (BYbes) molecular weight cut off membraneand centrifugation at 4000×g in a swing out rotor. Concentrated sampleswere applied to either a XK16/60 or XK26/60 Superdex200 column (GEHealthcare) equilibrated in PBS, pH7.4. The columns were developed withan isocratic gradient of PBS, pH7.4 at either 1 ml/min or 2.6 ml/minrespectively. Fractions were collected and analysed by size exclusionchromatography on a TSK gel G3000SWXL; 5 μm, 7.8×300 mm column developedwith an isocratic gradient of 0.2M phosphate, pH7.0 at 1 ml/min, withdetection by absorbance at 280 nm. Selected monomer fractions werepooled and concentrated to >1 mg/ml using an Amicon Ultra-15concentrator with a 10 kDa molecular weight cut off membrane andcentrifugation at 4000×g in a swing out rotor. Final samples wereassayed; for concentration by A280 Scanning UV-visible spectrophotometer(Cary 50Bio); for % monomer by size exclusion chromatography on a TSKgel G3000SWXL; 5 μm, 7.8×300 mm column developed with an isocraticgradient of 0.2 M phosphate, pH7.0 at 1 ml/min, with detection byabsorbance at 280 nm; by reducing and non-reducing SDS-PAGE run on 4-20%Tris-Glycine 1.5 mm gels (Novex) at 50 mA (per gel) for 53 minutes; andfor endotoxin by Charles River's ENDOSAFE® Portable Test System withLimulus Amebocyte Lysate (LAL) test cartridges.

Functional Assays

Activation Marker Assay:

Antigen 2-specific Fab′-Y and Antigen 3-specific Fab′-X, were incubatedtogether for 60 minutes (in a 37° C. and 5% CO₂ environment) atequimolar concentration. The combinations were titrated from a startingmolarity of 100 nM, in 1:4 serial dilutions. Antigen 2 and 3-specificBYbe was also titrated from a starting molarity of 100 nM, in 1:4 serialdilutions. In V-bottomed 96 well plates, 1.5×10⁵ PBMC were added towells, to which were added titrated Fab′-X and Fab′-Y combinations ortitrated BYbe. The Fab′-X and Fab′-Y combinations or BYbe were incubatedwith cells for a further 90 minutes. After this time B cells wereactivated by the addition of 25 μg/mL of goat F(ab′)2 anti-human IgM(Southern Biotechnology) for 24 hours at 37° C. plus 5% CO₂.

To the wells were added 100 μL ice-cold FACS buffer (PBS+1% BSA+0.1%NaN₃+2 mM EDTA), the plates were sealed and covered with wet-ice forapproximately 15 minutes, before centrifuging at 500×g for 5 minutes at4° C. Excess supernatant was discarded from the cell pellets and theplates shaken at 2000 rpm for 30 seconds.

Cells were then stained with a cocktail of fluorescently labelledanti-CD19, anti-CD20 and anti-CD71, anti-CD40 and anti-CD86 antibodies(BD Biosciences). Plates were shaken briefly and incubated for 1 hour onwet-ice in the dark. After this time plates were washed twice andresuspended in 20 μL of FACS buffer. Cellular expression of CD19, CD20and CD71, CD40 and CD86 was measured using an Intellicyt IQUE® Screenerflow cytometer. Using the data analysis software package FORECYT™(Intellicyt) B cells were identified as distinct from other cellpopulations and the geometric mean of CD71, CD40 and CD86 levels werecalculated for each well. All data was then expressed as the percentageinhibition of the maximal response (anti-IgM only) minus the background(cells only).

PhosFlow Assay:

Antigen 2-specific Fab′-Y and Antigen 3-specific Fab′-X, were incubatedtogether for 60 minutes (in a 37° C. and 5% CO₂ environment) atequimolar concentration. The combinations were titrated from a startingmolarity of 100 nM, in 1:4 serial dilutions. Antigen 2 and Antigen3-specific BYbe was also titrated from a starting molarity of 100 nM, in1:4 serial dilutions. In V-bottomed 96 well plates, 5.0×10⁴ PBMC wereadded to wells, to which were added titrated Fab′-X and Fab′-Ycombinations or titrated BYbe. The Fab′-X and Fab′-Y combinations orBYbe were incubated with cells for a further 90 minutes. After this timeB cells were activated by the addition of 25 μg/mL of goat F(ab′)2anti-human IgM (Southern Biotechnology) for 15 minutes at 37° C. plus 5%CO₂. The signalling reaction was then halted by adding an equal volumeof Cytofix buffer (BD Biosciences). Plates were then left at roomtemperature for 15 minutes before centrifugation at 500×g for 5 minutes.Excess supernatant was discarded from the cell pellet which wasresuspended in FACS buffer (PBS+1% BSA+0.01% NaN₃+2 mM EDTA) and washedonce more. Cells were then resuspended in ice cold Perm Buffer III (BDBiosciences) for 30 minutes before being washed twice in flow buffer.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences) and anti-phosphorylated PLCγ2, anti-phosphorylated Aktand anti-phosphorylated p38 antibodies (BD Biosciences). Plates werethen resuspended and incubated for 1 hour at room temperature in thedark. After this time plates were washed a further two times andresuspended in 20 μL of FACS buffer. Cellular expression of CD20 andphospho-PLCγ2, phospho-Akt and phospho-p38 were measured using anIntellicyt IQUE® flow cytometer. Using the data analysis softwarepackage FORECYT™ (Intellicyt) B cells were identified as distinct fromother cell populations and the geometric mean of PLCγ2, Akt and p38levels were calculated for each well. All data was then expressed as thepercentage inhibition of the maximal response (anti-IgM only) minus thebackground (cells only).

Results

PhosFlow Assay:

The data in FIG. 34 show that targeting antigen 3 and antigen 2 eitherin the Fab-Kd-Fab or BYbe format can inhibit phosphorylated PLCγ2 inB-cells stimulated with anti-IgM. The data in FIG. 35 show thattargeting antigen 3 and antigen 2 either in the Fab-Kd-Fab or BYbeformat can inhibit phosphorylated P38 in B-cells stimulated withanti-IgM. The data in FIG. 36 show that targeting antigen 3 and antigen2 either in the Fab-Kd-Fab or BYbe format can inhibit phosphorylated Aktin B-cells stimulated with anti-IgM.

Activation Marker Assay:

As can be seen in FIG. 37, the data show that targeting antigen 3 andantigen 2 either in the Fab-Kd-Fab or BYbe format can inhibit CD71expression on B-cells stimulated with anti-IgM. The data in FIG. 38 showthat targeting antigen 3 and antigen 2 either in the Fab-Kd-Fab or BYbeformat can inhibit CD40 expression on B-cells stimulated with anti-IgM.The data in FIG. 39 show that targeting antigen 3 and antigen 2 eitherin the Fab-Kd-Fab or BYbe format can inhibit CD86 expression on B-cellsstimulated with anti-IgM

Example 16—Comparison of the Activity of Antigen 2 Plus Antigen 3Co-Targeting in a Molecularly Linked Bispecific Bybe Format with theFurther Addition of an Anti-Albumin Binding Domain for Extension of InVivo Half-Life

Introduction:

To check that target pair activity identified in the Fab-Kd-Fabheterodimerically tethered screening complex could translate to similardesired activity in a potential therapeutic molecularly linked formatwith an anti-albumin targeted in vivo half-life extension, ananti-albumin antibody fragment was fused to the light chain of theantigen 3 Fab of the BYbe format described in Example 15. Antigen 2specificity (VR4447) and antigen 3 specificity (VR4130 and VR4126) weregenerated in a Bybe format with and without addition of an anti-albuminfragment (VR0645).

Description of Constructs Used in this Experiment.

Construct Name Fab Specificity Heavy Chain Light Chain VR4447/VR4126BYbe Antigen 2 Antigen 3 None VR4447/VR4126/VR645) Antigen 2 Antigen 3Albumin BYbe/Albumin VR4447/VR4130 BYbe Antigen 2 Antigen 3 NoneVR4447/VR4130/VR645) Antigen 2 Antigen 3 Albumin BYbe/Albumin

Methods

Purification of BYbes for Functional Screening:

The functional screening BYbe (Fab-dsscFv [scFv off C-terminus of Fabheavy chain]) format was purified as follows. Clarified cell culturesupernatants from standard expiHEK or CHO expression were 0.22 μmsterile filtered. The filtered supernatants were loaded at 2 ml/min onto50 ml GammabindPlus Sepharose XK26 columns (GE Healthcare) equilibratedin PBS pH7.4 (Sigma Aldrich Chemicals). After loading the columns werewashed with PBS pH7.4 and then eluted with 0.1M Glycine/HCl. pH 2.7. Theelution was followed by absorbance at 280 nm, the elution peakcollected, and then neutralised with 1/25^(th) volume of 2 M Tris/HClpH8.5. The neutralised samples were concentrated using Amicon Ultra-15concentrators with either a 10 kDa or 30 kDa molecular weight cut offmembrane and centrifugation at 4000×g in a swing out rotor. Concentratedsamples were applied to either a XK16/60 or XK26/60 Superdex 200 column(GE Healthcare) equilibrated in PBS, pH7.4. The columns were developedwith an isocratic gradient of PBS, pH7.4 at either 1 ml/min or 2.6ml/min respectively. Fractions were collected and analysed by sizeexclusion chromatography on a TSK gel G3000SWXL; 5 μm, 7.8×300 mm columndeveloped with an isocratic gradient of 0.2 M phosphate, pH 7.0 at 1ml/min, with detection by absorbance at 280 nm. Selected monomerfractions were pooled and concentrated to >1 mg/ml using an AmiconUltra-15 concentrator with a 10 kDa or 30 kDa molecular weight cut offmembrane and centrifugation at 4000×g in a swing out rotor. Finalsamples were assayed; for concentration by A280 Scanning UV-visiblespectrophotometer (Cary 50Bio); for % monomer by size exclusionchromatography on a TSK gel G3000SWXL; 5 μm, 7.8×300 mm column developedwith an isocratic gradient of 0.2 M phosphate, pH7.0 at 1 ml/min, withdetection by absorbance at 280 nm; by reducing and non-reducing SDS-PAGErun on 4-20% Tris-Glycine 1.5 mm gels (Novex) at 50 mA (per gel) for 53minutes; and for endotoxin by Charles River's ENDOSAFE® Portable TestSystem with Limulus Amebocyte Lysate (LAL) test cartridges.

100 nM of each construct purified protein were pre-incubated with humanPBMC derived from five separate donors for 60 min at 37 degree C./5% CO₂in RMPI 1640 media plus 10% foetal bovine serum and 2 mM Glutamax (R10media). After 60 min cells were stimulated with 25 ug/ml of a goatanti-IgM antibody designed to stimulate B cells only. 24 hours laterplates were placed on ice to halt any further cell activation beforewashing once with ice cold flow cytometry buffer (PBS+1% BSA+0.01%NaN₃). All supernatant was removed and cell pellets resuspended. Cellswere placed on ice and a cocktail of anti-CD19, -CD20, -CD27, -CD71 andCD86 antibodies added. Cells were incubated for 60 min before washingtwice in flow cytometry buffer. Data on the binding of anti-CD27, -CD71and CD86 to CD19/CD20 positive B cells was generated using an iQUE highthroughput flow cytometer. Forecyt software was used to generatehistograms and derive geometric mean intensity readings for the bindingof anti-CD27, -CD71 and CD86 antibodies to B cells. This data wasimported into Excel and percentage inhibition values generated for eachcombination. The data was then imported into Graphpad Prism and box andwhisker charts generated for each combination with the mean indicated bya ‘+’.

FIG. 40 shows the inhibition of CD27 expression on B cells induced byVR4447/VR4126 BYbe and VR4447/VR4126/VR645 BYbe/Albumin. Across the fivedonors tested both showed consistently similar levels of inhibition ofanti-IgM induced CD27. FIG. 41 shows the inhibition of CD71 expressionon B cells induced by VR4447/VR4126 BYbe and VR4447/VR4126/VR645BYbe/Albumin. Across the five donors both showed consistently similarlevels of inhibition of anti-IgM induced CD71. FIG. 42 shows theinhibition of CD86 expression on B cells induced by VR4447/VR4126 BYbeand VR4447/VR4126/VR645 BYbe/Albumin. Across the five donors both showedconsistently similar levels of inhibition of anti-IgM induced CD86.

GCN4(7P14P) sequences SEQ ID NO: 1ASGGGRMKQLEPKVEELLPKNYHLENEVARLKKLVGERHHHHHHwherein the amino acids in bold are optionaland the amino acids in italics are the linking sequence SEQ ID NO: 2GCTAGCGGAGGCGGAAGAATGAAACAACTTGAACCCAAGGTTGAAGAATTGCTTCCGAAAAATTATCACTTGGAAAATGAGGTTGCCAGATTAAAGAAATTAGTTGGCGAACGCCATCACCATCACCATCAC 52SR4 ds scFv sequence SEQ ID NO: 3DAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTVLGGGGGSGGGGSGGGGSGGGGSDVQLQQSGPGLVAPSQSLSITCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSAAAHHHH HHEQKLISEEDL-SEQ ID NO: 4 GATGCGGTGGTGACCCAGGAAAGCGCGCTGACCAGCAGCCCGGGCGAAACCGTGACCCTGACCTGCCGCAGCAGCACCGGCGCGGTGACCACCAGCAACTATGCGAGCTGGGTGCAGGAAAAACCGGATCATCTGTTTACCGGCCTGATTGGCGGCACCAACAACCGCGCGCCGGGCGTGCCGGCGCGCTTTAGCGGCAGCCTGATTGGCGATAAAGCGGCGCTGACCATTACCGGCGCGCAGACCGAAGATGAAGCGATTTATTTTTGCGTGCTGTGGTATAGCGACCATTGGGTGTTTGGCTGCGGCACCAAACTGACCGTGCTGGGTGGAGGCGGTGGCTCAGGCGGAGGTGGCTCAGGCGGTGGCGGGTCTGGCGGCGGCGGCAGCGATGTGCAGCTGCAGCAGAGCGGCCCGGGCCTGGTGGCGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTCTCCTGACCGATTATGGCGTGAACTGGGTGCGCCAGAGCCCGGGCAAATGCCTGGAATGGCTGGGCGTGATTTGGGGCGATGGCATTACCGATTATAACAGCGCGCTGAAAAGCCGCCTGAGCGTGACCAAAGATAACAGCAAAAGCCAGGTGTTTCTGAAAATGAACAGCCTGCAGAGCGGCGATAGCGCGCGCTATTATTGCGTGACCGGCCTGTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGCGGCCGCCCATCACCATCACCATCACGAACAGAAACTGATTAGCGAAGAAGATCTGTAATAG

1. A bispecific protein complex having the formula A-X:Y-B wherein: A-Xis a first fusion protein; Y-B is a second fusion protein; : is abinding interaction between X and Y to form a bispecific protein complexbetween the first and second fusion proteins; A is a first proteincomponent of the bispecific protein complex selected from a Fab or Fab′fragment; B is a second protein component of the bispecific proteincomplex selected from a Fab or Fab′ fragment; X is a first bindingpartner of a binding pair independently selected from an antigen or anantibody or binding fragment thereof, wherein when X is an antigen, Y isan antibody or binding fragment thereof specific to the antigenrepresented by X; and Y is a second binding partner of the binding pairindependently selected from an antigen or an antibody or a bindingfragment thereof, wherein when Y is an antigen, X is an antibody orbinding fragment thereof specific to the antigen represented by Y;wherein A and B bind to two different epitopes on an antigen, or bindtwo different antigens, and wherein X or Y is a VHH specific to thepeptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1,and wherein X or Y is a peptide GCN4 having SEQ ID NO:1 or amino acids 1to 38 of SEQ ID NO:1.
 2. A bispecific protein complex according to claim1, wherein A is a Fab fragment.
 3. A bispecific protein complexaccording to claim 1, wherein B is a Fab fragment.
 4. A bispecificprotein complex according to claim 1, wherein X is fused directly or viaa linker, to the C-terminal of the heavy chain in the Fab or Fab′fragment.
 5. A bispecific protein complex according to claim 1, whereinY is fused directly or via a linker, to the C-terminal of the heavychain in the Fab or Fab′ fragment.
 6. (canceled)
 7. (canceled) 8.(canceled)
 9. A bispecific protein complex according to claim 1, whereinthe binding affinity between X and Y is 5 nM or stronger.
 10. Abispecific protein complex according to claim 9, wherein the bindingaffinity between X and Y is 900 pM or stronger.
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. The bispecific protein complex accordingto claim 1, wherein A and/or B is specific for an antigen selected fromthe group comprising: cell surface receptors, co-stimulatory molecules,checkpoint inhibitors, natural killer cell receptors, Immunoglobulinreceptors, TNFR family receptors, B7 family receptors, adhesionmolecules, integrins, cytokine/chemokine receptors, GPCRs, growth factorreceptors, kinase receptors, tissue-specific antigens, cancer antigens,pathogen recognition receptors, complement receptors, hormone receptors,cytokines, chemokines, leukotrienes, growth factors, hormones, enzymes,and ion channels.
 15. A composition comprising the bispecific proteincomplex defined in claim
 1. 16. A method of treating a subject in needthereof comprising administering a bispecific protein complex accordingto claim 1 to the subject.
 17. A method of detecting synergisticbiological function in a bispecific protein complex according to claim 1comprising testing the bispecific protein complex in one or morefunctional assays.
 18. A method for detecting synergistic biologicalfunction in a heterodimerically-tethered bispecific protein complex offormula A-X:Y-B, : is a binding interaction between X and Y to form abispecific protein complex between the first and second fusion proteins,A and B are protein components of the bispecific in the form of fusionproteins with X and Y respectively, said method comprising the steps of:(i) testing for activity in a functional assay for part or all of amultiplex comprising at least one heterodimerically-tethered bispecificprotein complex; and (ii) analysing the readout(s) from the functionalassay to detect synergistic biological function in theheterodimerically-tethered bispecific protein complex; and wherein A andB bind to two different epitopes on an antigen, or bind two differentantigens, and wherein X or Y is a VHH specific to the peptide GCN4having SEQ ID NO:1 or amino acids 1 to 38 of SEQ ID NO:1, and wherein Xor Y is a peptide GCN4 having SEQ ID NO:1 or amino acids 1 to 38 of SEQID NO:1.
 19. A method according to claim 18, wherein the multiplex is inthe form a grid.
 20. A method according to claim 18 wherein themultiplex comprises at least two heterodimerically-tethered bispecificprotein complexes.
 21. A method according to claim 18, wherein theheterodimerically tethered bispecific protein complex(es) do not containan Fc region.
 22. A method according to claim 18, wherein A isindependently selected from a Fab fragment, a Fab′ fragment, a VHH, aVH, a VL and a scFv.
 23. A method according to claim 22, wherein A is aFab or Fab′ fragment.
 24. A method according to claim 18, wherein B isindependently selected from an antibody, a Fab fragment, a Fab′fragment, a VHH, a VH, a VL and a scFv.
 25. A method according to claim24, wherein B is a Fab or Fab′ fragment.
 26. A method according to claim24, wherein X is fused directly or via a linker, to the C-terminal of aheavy chain in the antibody, Fab or Fab′ fragment.
 27. A methodaccording to claim 24, wherein Y is fused directly or via a linker tothe C-terminal of a heavy chain in the antibody, Fab or Fab′ fragment.28. (canceled)
 29. (canceled)
 30. (canceled)
 31. A method according toclaim 18 wherein the binding affinity between X and Y is 5 nM orstronger.
 32. A method according to claim 31, wherein the bindingaffinity of between X and Y is 900 pM or stronger.
 33. (canceled) 34.(canceled)
 35. (canceled)
 36. A method according to claim 18, wherein Aand/or B is specific for an antigen selected from the group comprising:cell surface receptors, co-stimulatory molecules, checkpoint inhibitors,natural killer cell receptors, Immunoglobulin receptors, TNFR familyreceptors, B7 family receptors, adhesion molecules, integrins,cytokine/chemokine receptors, GPCRs, growth factor receptors, kinasereceptors, tissue-specific antigens, cancer antigens, pathogenrecognition receptors, complement receptors, hormone receptors,cytokines, chemokines, leukotrienes, growth factors, hormones or enzymesor ion channels.
 37. A method according to claim 18, wherein theheterodimerically tethered bispecific protein complexes are not purifiedprior to testing.
 38. A method according to claim 37, wherein the A-Xand Y-B fusion proteins are expressed transiently and not purifiedbefore being mixed in a 1:1 molar ratio to generate eachheterodimerically tethered bispecific protein complex.